Low intensity focused ultrasound for treating cancer and metastasis

ABSTRACT

Systems and methods for treating cancer and for preventing metastasis using low intensity focused ultrasound in combination with an anti-cancer therapy are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Phase entry of InternationalApplication No. PCT/US2016/035440, filed Jun. 2, 2016, which claims thebenefit of U.S. Provisional Application No. 62/170,378, filed Jun. 3,2015, and of U.S. Provisional Application No. 62/204,312, filed Aug. 12,2015, the contents of each are incorporated herein by reference in theirentireties.

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 16, 2016, isnamed 52650704831_SL.txt and is 5,178 bytes in size.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbersEB009040 and AI059738 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referred to inbrackets. Full citations for these references may be found at the end ofthe specification. The disclosures of these publications, and of allpatents, patent application publications and books referred to herein,are hereby incorporated by reference in their entirety into the subjectapplication to more fully describe the art to which the subjectinvention pertains.

Immune responses against cancer cells are frequently hampered by theimmunosuppressive nature of the tumor microenvironment, which is alsoresponsible for hindering the efficacy of cancer immunotherapy (1, 2).Several mechanisms have been identified underlying the ability of tumorsto generate an immunosuppressive environment, including secretion ofcytokines or other factors with inhibitory activity (3-5), recruitmentof regulatory T cells and myeloid-derived suppressor cells (6-9),increased expression of ligands for co-inhibitory receptors (10-13) orinhibition of dendritic cell maturation (14, 15). As a consequence ofthose mechanisms T cells are often rendered unresponsive to tumorantigens (15). Induction of a hyporesponsive state to tumor antigensoccurs both in CD4+ and CD8+ T cell populations and is often responsiblefor the inability of the adaptive immune system to mount an efficientanti-tumor response (16-18). Decreased T cell responses to tumorantigens occur in both solid and hematological tumors and appear to becaused by inefficient presentation of antigens by dendritic cells, whichresults in the preferential activation of tolerogenic programs of geneexpression that are dependent on the transcription factors NFAT and Egr2(18-21). The important role of this process of tumor-induced T cellhyporesponsiveness is underscored by the fact that genetic mouse modelswhere the induction of this tolerogenic gene expression program isprevented result in enhanced anti-tumor T cell responses and control oftumor growth (19, 21).

Treatment of localized tumors by focused ultrasound (FUS) is an imageguided minimally invasive therapy that uses a range of input energy forin situ tumor ablation (22, 23). The application of FUS to biologicaltissues is associated with the generation of thermal and cavitationeffects, causing changes in target cell physiology, depending on theenergy delivered. High intensity focused ultrasound (HIFU) has been usedclinically to thermally ablate localized tumors (23-26). The substantialthermal energy generated by that modality of FUS treatment causes rapidcoagulative necrosis of the tissue at the targeted focal spots. Thoughseveral studies have reported some immunomodulatory effects, includingincreased lymphocyte infiltration, generation of IFNγ producingtumor-specific T cells in lymphoid organs and dendritic cell maturationand migration into tumors (26, 27), the thermally induced coagulativenecrosis resulting from HIFU treatment can also attenuate the release ofimmunostimulatory molecules within the tumor microenvironment. Thus,although able to halt the progression of established primary tumors,HIFU might fail to protect against local and distant metastases arisingfrom the surviving tumor cells.

The present invention provides improved tumor and cancer treatmentsemploying low intensity focused ultrasound, and methods of inducingchemosensitization and increasing the efficacy of cancer otherapytreatments.

SUMMARY OF THE INVENTION

A method is provided for increasing the efficacy of a chemotherapy in asubject comprising administering to the subject (i) an amount of lowintensity focused ultrasound (LOFU) and (ii) an amount of achemotherapeutic drug, wherein the chemotherapeutic drug effectsendoplasmic reticulum (ER) stress and/or unfolded protein response (UPR)in a tumor cell, wherein the amounts (i) and (ii) together aresufficient to increase the efficacy of the chemotherapy.

Also provided is a method of increasing the efficacy of a chemotherapyin a predetermined volume of tissue in a subject which volume is lessthan the whole subject, comprising (i) administering to the subject anamount of a chemotherapeutic drug, wherein the chemotherapeutic drugeffects endoplasmic reticulum (ER) stress and/or unfolded proteinresponse (UPR) in a tumor cell and (ii) administering to thepredetermined volume of tissue in a subject an amount of low intensityfocused ultrasound (LOFU), wherein the amounts of (i) and (ii) togetherare sufficient to increase efficacy of the chemotherapy within thepredetermined volume of tissue.

A method of treating a tumor in a subject is provided, wherein the tumoris resistant to a chemotherapeutic drug, comprising:

receiving identification of the subject as having a tumor resistant to aspecified chemotherapeutic drug;

administering (i) an amount of low intensity focused ultrasound (LOFU)and (ii) an amount of the specified chemotherapeutic drug,

wherein the amounts (i) and (ii) together are sufficient to treat thetumor.

Also provided is a method of treating a chemoresistant tumor in asubject, wherein the tumor has become chemoresistant to a previouslyadministered chemotherapeutic drug, comprising:

administering to the subject (i) an amount of low intensity focusedultrasound (LOFU) and (ii) an amount of the chemotherapeutic drug,

wherein the amounts (i) and (ii) together are sufficient to treat thechemoresistant tumor.

Acoustic priming therapy (APT) systems according to various exemplaryembodiments of the present invention comprise transducers configured toproduce acoustic power between 10 and 1000 W/cm² spatial peak temporalaverage intensity (Ispta) in a treatment zone, wherein the frequency ofthe ultrasound is in the range of 0.01 to 10 MHz, the mechanical indexis less than 4 and the ultrasound is applied continuously from a time inthe range of 0.5 to 5 seconds for any particular volume in the treatmentzone. Such treatments are identified herein as acoustic priming therapy(APT) treatments.

An acoustic priming therapy device according to an exemplary embodimentof the present invention comprises: a control system that generates afrequency waveform; and one or more transducers each configured toproduce ultrasonic beams based on the frequency waveform with a peakfrequency in the range of 0.5 to 5 MHz and an acoustic output intensityof between 20 and 1000 W/cm².

In an exemplary embodiment, each transducer is configured to producecolumnated ultrasound such that the beam profile waist at −3 dB is notless than 5 mm in a treatment zone.

In an exemplary embodiment, two or more transducers can be operatedsequentially or simultaneously and produce ultrasound of average spatialpeak 250 J/cm² in a treatment zone during a treatment period.

In an exemplary embodiment, the transducers are operated in continuousmode wherein ultrasound is produced in a treatment zone for a treatmentperiod in the range of 0.1 to 10 seconds.

A system according to an exemplary embodiment of the present inventioncomprises: an acoustic priming therapy device comprising: a controlsystem that generates a frequency waveform; and one or more transducersconfigured to produce ultrasound based on a frequency waveform between 1and 1000 W/cm² spatial peak temporal average acoustic output intensity(I_(spta)) in a treatment zone, wherein the ultrasound is appliedcontinuously for a time in the range of 0.5 to 5 seconds, and whereinultrasound frequency is in the range of 0.01 to 10 MHz; a radiotherapytreatment machine; and a control system operatively configured tocontrol the acoustic priming therapy device and the radiotherapytreatment machine so that a first amount of the ultrasound and a secondamount of radiotherapy are administered to a subject, wherein the firstand second amounts together are sufficient to treat a tumor in thesubject.

A system according to an exemplary embodiment of the present inventioncomprises: an acoustic priming therapy device comprising: a controlsystem that generates a frequency waveform; and one or more transducersconfigured to produce ultrasound based on a frequency waveform between 1and 1000 W/cm² spatial peak temporal average acoustic output intensity(Ispta) in a treatment zone, wherein the ultrasound is appliedcontinuously for a time in the range of 0.5 to 5 seconds, and whereinultrasound frequency is in the range of 0.01 to 10 MHz; the acousticpriming therapy device for use in combination with chemotherapy so thata first amount of the ultrasound and a second amount of the chemotherapyare administered to a subject, wherein the first and second amountstogether are sufficient to treat a tumor in the subject.

A system according to an exemplary embodiment of the present inventioncomprises: an acoustic priming therapy device comprising: a controlsystem that generates a frequency waveform; and one or more transducersconfigured to produce ultrasound based on a frequency waveform between 1and 1000 W/cm² spatial peak temporal average acoustic output intensity(Ispta) in a treatment zone, wherein the ultrasound is appliedcontinuously for a time in the range of 0.5 to 5 seconds, and whereinultrasound frequency is in the range of 0.01 to 10 MHz; the acousticpriming therapy device for use in combination with immunotherapy so thata first amount of the ultrasound and a second amount of theimmunotherapy are administered to a subject, wherein the first andsecond amounts together are sufficient to treat a tumor in the subject.

A method of treating a tumor in a subject is provided comprisingadministering to the subject (i) an amount of low intensity focusedultrasound (LOFU) and (ii) an amount of chemotherapy or an amount ofradiotherapy or an amount of immunotherapy, wherein the amounts of (i)and (ii) together are sufficient to treat a tumor.

Also provided is a method of inhibiting metastasis of a tumor in asubject, comprising administering to a subject having a tumor an amountof low intensity focused ultrasound (LOFU) and an amount of aradiotherapy, wherein the amounts together are sufficient to treat atumor.

Also provided is a method of reducing the effective dose of ananti-cancer chemotherapy required to treat a tumor in a subjectcomprising administering to the subject undergoing the anti-cancerchemotherapy an amount of low intensity focused ultrasound (LOFU)sufficient to reduce the effective dose of the anti-cancer chemotherapyrequired to treat a tumor.

Also provided is a method of sensitizing a tumor in a subject to anamount of an anti-cancer therapy the method comprising administering tothe subject, prior to or during the course of the anti-cancer therapy,an amount of an acoustic priming therapy effective to sensitize a tumorin a subject to an amount of an additional anti-cancer therapy modality.

A method of treating a tumor in a subject is provided comprisingadministering to the subject (i) an amount of low intensity focusedultrasound (LOFU) with LOFU herein indicating an exemplary embodiment ofan ultrasound configuration used in an APT system, and (ii) an amount ofchemotherapy or an amount of radiotherapy or an amount of immunotherapy,wherein the amounts of (i) and (ii) together are sufficient to treat atumor.

Also provided is a method of inhibiting metastasis of a tumor in asubject, comprising administering to a subject having a tumor an amountof low intensity focused ultrasound (LOFU) and an amount of aradiotherapy, wherein the amounts together are sufficient to inhibitmetastasis of a tumor in a subject.

Also provided is a method of reducing the effective dose of ananti-cancer chemotherapy required to treat a tumor in a subjectcomprising administering to the subject undergoing the anti-cancerchemotherapy an amount of low intensity focused ultrasound (LOFU)sufficient to reduce the effective dose of the anti-cancer chemotherapyrequired to treat a tumor.

Also provided is a method of sensitizing a tumor in a subject to anamount of an anti-cancer therapy the method comprising administering tothe subject, prior to or during the course of the anti-cancer therapy,an amount of low intensity focused ultrasound (LOFU) effective tosensitize a tumor in a subject to an amount of an anti-cancer therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1F. Melanoma tumors suppress cytokine output of CD4+ T cells:1A-B: C57Bl/6 mice were challenged in the lumbar flanks with 3×10⁵B16-F1 melanoma cells. Tumors were allowed to grow to 7-8 mm³ in size.CD4+ T cells were isolated from the tumor DLN and distal contralateralNDLN, and stimulated with anti-CD3 and anti-CD28 antibodies. IL-2 andIFNγ were measured by ELISA. CD4+ T cells from tumor-free mice were usedas controls. 1C-D: OTII mice were challenged with 3×10⁵ B16-F1-OVAmelanoma cells as described. T cells were stimulated with OVA323-339peptide-loaded splenocytes and IL-2 and IFNγ production measured byELISA. 1E-F. B16-F1 cells were used to induce tumors in Tyrp1 mice asdescribed above. Isolated CD4+ T cells were stimulated with anti-CD3 andanti-CD28 antibodies and IL-2 and IFNγ production determined by ELISA.Graphs show mean+SEM from 4 (1A-B) or 3 (1C-F) independent experiments.Results are shown as mean+SEM from 3-5 mice for each experiment. Datawere analyzed using ANOVA with a Tukey post-test (***P<0.01; **P<0.01;*P<0.05).

FIG. 2A-2D. Treatment of melanoma tumors with LOFU overcomes tumorinduced CD4+ T cell tolerance: 2A-B. Tumors were induced in C57Bl/6 miceby s.c. injection of 3×10⁵ B16-F1 melanoma cells in the lumbar flank.Tumors were left untreated or treated with LOFU. Thirty-six hours afterFUS treatment, CD4+ T cells were isolated from tumor DLN or NDLNs andstimulated with anti-CD3 and anti-CD28 antibodies. IL-2 and IFNγproduction was assessed by ELISA. The results (total cytokine productionand ratio of the levels of cytokines produced by T cells from NDLN andDLN in each group) are presented as mean+SEM from 3 different mice percondition. Differences between cytokine production of DLN T cells inuntreated or treated mice were analyzed using a 2-tailed t test(*P<0.05). 2C. Mice were challenged with 3×10⁵ B16 melanoma cells toinduce tumors. Following tumor development total RNA samples wereextracted from CD4+ T cells isolated from the DLN and NDLN oftumor-bearing mice, and tumor-free control mice. Expression ofanergy-associated genes was measured by quantitative RT-PCR. The resultsare shown as fold induction of gene expression in the DLN or NDLNresident T cells in tumor bearing mice compared to T cells isolated fromtumor-free mice. The data represent mean+SEM from 3 independentexperiments. 2D. B16-F1 melanoma tumors were induced in Tyrp1 mice thatwere then left untreated or treated with LOFU. The expression ofdifferent anergy-associated genes was measured by RT-PCR in CD4+ T cellsisolated from the DLNs and NDLNs. Expression of the anergy-associatedgenes is presented as fold induction (mean+SEM from 5 independentexperiments) over the values obtained in T cells from Tyrp1 mice bearingno tumor.

FIG. 3A-3B. Lysates from LOFU-treated B16-F1 melanoma tumors can reversethe hyporesponsive state of anergic T cells 3A. Naïve CD4+ T cells wereisolated from spleens and lymph nodes of Tyrp1 mice, and differentiatedinto TH1 cells. Cells were then either left untreated or treated withanti-CD3 alone for 16 hours to induce anergy. Cells were then rested for72 hours in strict absence of IL-2 and re-stimulated with anti-CD3 andanti-CD28 antibodies. IL-2 levels were measured by ELISA. The resultsare shown as mean+SEM from 2 independent experiments. 3B. CD11c+dendritic cells were isolated from spleens of tumor-free Tyrp1 mice.Anergic TH1 cells generated from Tyrp1 mouse-derived CD4+ T cells asdescribed in (3A) were co-cultured with the dendritic cells and tumorlysates derived from untreated or LOFU-treated B16-F1 melanoma tumors.Supernatants were collected after 24 hours and assayed for IL-2 byELISA. Results are shown as mean+SEM from 2 independent experiments with3 independent sets of tumor lysates used in each experiment. Data wereanalyzed using ANOVA with a Tukey post-test (**P<0.01).

FIG. 4A-4D. FUS treatment causes changes in expression and cellulardistribution of Hsp70 and calreticulin in B16-F1 melanoma cells. 4A.Total DLNs resident cells from untreated and LOFU-treated B16-F1melanoma-bearing mice were isolated and immunostained for CD11c to gatedendritic cells. Surface expression of B7.1, B7.2 and MHCII was thenassessed by flow cytometry. Appropriate isotype controls were used foreach primary antibody. Representative histograms are shown. 4B.Representative FACS dot plot of B16 tumor cell suspension obtained fromuntreated or LOFU treated mice were stained with a viability marker(Live/dead Mk). Relative quantification of dead cells is reported. Boxand arrow indicate dead cells (Live/dead MK+). 4C. Immunofluorescencestaining of B16-F1 tumor tissues isolated from untreated mice or frommice treated with LOFU. Tissue sections were stained with antibodies todetect calreticulin or Hsp70 and TRP1. Nuclei were stained with DAPI.Magnification 60×. 4D. Cells from tumors of LOFU treated mice anduntreated mice were stained for CD45 and for the expression of TRP1.CD45-TRP1+B16 cells were then analyzed for the expression of Hsp70. Arepresentative histogram is shown. Gates and arrows indicate theselected population for the analysis.

FIG. 5A-5B. FUS treatment of melanoma tumors potentiate dendriticcell-mediated priming of CD4+ T cells: 5A. CD11c+ splenic dendriticcells were purified from C57Bl/6 mice and co-cultured with respondernaïve CD4+ T cells isolated from OT-II mice. B16-F1-OVA melanoma tumorlysates were prepared from untreated or LOFU treated tumor-bearing miceand added to the respective cultures to drive dendritic cell mediated Tcell stimulation. In separate samples exogenous OVA₃₂₃₋₃₃₉ peptide wasalso added along with tumor lysates. Supernatants were collected after24 hours, and IL-2 production was assessed by ELISA. The results areshown as mean+SEM from 4 independent experiments and analyzed withone-way ANOVA followed by a Tukey posttest (*P<0.05; ***P<0.001; n.s.,not significant). 5B. B16-F1 melanoma tumors were left untreated ortreated with LOFU. Tumor DLN were isolated and depleted of T cells. DLNcells were then co-cultured with naïve Tyrp1 CD4+ T cells and stimulatedwith B16 melanoma tumor lysates obtained from in vitro cultures.Supernatants were collected 24 hours later and analyzed for IL-2 levelsby ELISA. The data is shown as mean+SEM from 3 independent experiments.Differences between cytokine production in cultures using DLN cells fromuntreated or LOFU-treated mice were analyzed using a 2-tailed t test(*P<0.05).

FIG. 6A-6D. FUS followed by hypofractionated IGRT results in T-cellmediated long term primary tumor control and reduced distal metastases:6A C57Bl/6 mice with 50 mm³ subcutaneous dorsal right hind limb tumorswere separated into one of four treatment groups: untreated, LOFU,hypofractionated IGRT, or LOGU+IGRT and tumor growth monitored for 62days or until primary tumor grew beyond 300 mm³. Graph shows mean±SEM oftumor volume from one of two representative experiments (3-5 mice pergroup). Data were analyzed with either one-way ANOVA followed by aBonferroni correction post test (before day 29) or by 2-tailed student ttest (after day 29). Significant differences (defined as P<0.05) betweenuntreated or LOFU-treated mice and IGRT or LOFU+IGRT treated miceoccurred after day 25, and between IGRT treated and LOFU+IGRT treatedmice after day 35. Individual graphs showing the distribution of tumorsize at specific days are also shown. 6B. Similar experiments as theones described in 6A were performed in BALB/c nude mice. No significantdifferences were observed among the different groups at any time point.6C. C57Bl/6 mice were monitored for primary tumorprogression/recurrence, defined as either recurrence reaching a volumeof 150 mm³ or the development of local metastasis to the popliteal oringuinal lymph nodes. In addition, animals that died spontaneously werescored as having recurrence or progression of disease. Recurrence freesurvival data was analyzed using the Mantel-Cox test. 6D. Lungs wereharvested from animals that either died spontaneously, requiredeuthanasia due to overwhelming tumor burden, or were sacrificed at theend of a two month long experiment. Lung metastasis were then measured.Lungs with nodules that fuse into plaques, or exceed 250 were deemed toonumerous to count and assigned a maximal value of 250. A representativespecimen is shown for each treatment group. The results are shown asmean+SEM, with n=3-5 mice per group, analyzed with a Kruskal-Wallistest, followed by Dunn's posttest. *P<0.05.

FIG. 7A-7G: LOFU induces UPR. 7A. LOFU increases the expression ofBip/Grp78 and EDEM mRNAs. Real Time-PCR analysis of RNA isolated fromLOFU-treated RM1 tumors showed 29.73±0.56 fold increase in Bip/Grp78 and9.27±1.18 fold increase in EDEM mRNA level compared to untreatedcontrol. 7B. LOFU increases the expression of IRE1α mRNA by 2.8±0.4folds. Real Time-PCR analysis demonstrates that LOFU induced increase inthe IRE1α expression did not alter with the 17AAG treatment. 7C. LOFUinduced the splicing of XBP1 mRNA. 17AAG treatment inhibits the splicingof XBP1. XBP1s, XBP1h, and XBP1u denote the spliced, hybrid, andun-spliced forms of XBP1, respectively. 7D-G. LOFU+17AAG combinationtherapy prolongs ER stress in RM1 tumor cells. Western blot and barchart showing that the expression of ERP78 (7D & 7E), ERP57 (7D & 7F),and ERp44 (7D & 7G) proteins was induced in combination treatment group.

FIG. 8A-8E: LOFU+17AAG activates pro-apoptotic pathways of UPR andinduces apoptosis in tumor cells. 8A & 8B. Western blot of pPERK (8A)and peIF2a (8B). LOFU+17AAG activates PERK by phosphorylation of PERK(pPERK), which further induces the phosphorylation of eIF2aphosphorylation (peIF2α). 8C. Real Time-PCR analysis of CHOP mRNA. Therewas a 25±1.3-fold increase in CHOP transcript in LOFU+17AAG treatedgroup, compared to control. 8D. Real Time-PCR array of RNA isolated fromLOFU+17AAGtreated tumors. Heat map analysis showed that LOFU+17AAGtreatment group increased the transcript level of apoptotic genesseveral folds compared to untreated control or LOFU groups. 8E. TUNELstaining. Immunohistochemical staining showed predominantly tunelpositive cells in LOFU+17AAG treatment group, compared to control orLOFU group. Note that 17AAG alone also induced apoptosis in tumor tissuethat was augmented by LOFU.

FIG. 9A-9C: LOFU+17AAG treatment inhibits Chaperone Mediated Autophagy(CMA) in RM1 tumor cells. (9A & 9B) Immunoblot analysis showed severalfold down-regulation of SMA marker LAMP2a expression level incombination treatment group. Treatment with either LOFU or 17AAGupregulates the LAMP2a expression level. (9A & 9C) Combination treatmentof LOFU and 17AAG did not alter the expression level of Beclin, amacroautophagy marker.

FIG. 10A-10C: Tumor growth retardation of murine and human prostatetumors after LOFU+17AAG treatment. 10A. Treatment schema. Palpabletumors were treated with LOFU every 3-4 days for five fractionsadministered over two weeks. Animals received 17AAG three times a weekduring this time. Tumors were harvested 24 hours after the last fractionof LOFU. 10B. RM1 tumor. In C57B16 mice, LOFU+17AAG combinationtreatment reduced RM1 tumor growth significantly (p<0.004), compared tocontrols. Note that either LOFU or 17AAG alone failed to control tumorssignificantly. LOFU sensitized the effects of a low dose (25 mg/kg ofbody weight) 17AAG. 10C. PC3 tumor. In BalbC nu/nu mice LOFU+17AAGcombination treatment showed significant reduction in PC3 tumor growth(p<0.007).

FIG. 11A-11F: LOFU+17AAG treatment reduces the expression of prostatecancer stem cell markers in RM1 cells. Flow cytometry of isolated RM1tumor cells showed significant decrease in SCA1 (11A & 11B), CD44 (11A &11C), CD133 (11A & 11D), and α2β1 integrin (11A & 11E) cell surfaceexpression on RM1 tumor cells after LOFU+17AAG treatment. (11F) qRT-PCRarray followed by heat map analysis showed that LOFU+17AAG combinationtreatment group down-regulates the mRNA levels of stem celltranscription factors.

FIG. 12 is a block diagram of an APT device according to an exemplaryembodiment of the present invention.

FIG. 13 is a block diagram of an APT device according to anotherexemplary embodiment of the present invention.

FIG. 14 is a block diagram of an APT device according to anotherexemplary embodiment of the present invention.

FIGS. 15-17 are block diagrams of APT devices according to exemplaryembodiments of the present invention including an integrated ultrasoundimaging device.

FIG. 18 is a perspective view of a transducer according to an exemplaryembodiment of the present invention.

FIG. 19 illustrates a positioning apparatus according to an exemplaryembodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is an ultrasound (US) therapy that delivers a reducedlevel of energy to a treatment zone compared to HIFU configurations. Inan exemplary embodiment, the treatment of a particular lesion volume isfor a short time (e.g. ˜1.5 sec) at 1 MHz continuous power, with tumortissue temperature elevated to less than about 45° C. This ultrasoundtreatment, generated using a concave transducer to focus the ulstrasoundin a treatment zone and herein termed “low energy non-ablative focusedultrasound” (LOFU), produces mild mechanical and thermal stress in tumorcells, while avoiding cavitation and coagulative necrosis both of whichresult in tissue damage. A non-ablative “sonic” stress response isinduced in the tumor that increases the expression of heat shockproteins without actually killing them directly. LOFU has the potentialto release immunomodulatory factors, including heat shock proteins (28,29), and can be effective in inducing tumor-specific immune activation(30, 31). Using a murine B16 melanoma tumor model, it is disclosed thatLOFU treatment reverses tumor-induced tolerance, resulting in increasedeffector cytokine production in tumor-antigen specific CD4+ T cells,which appears to be caused by the release of immunogenic molecules bythe tumor cells. Also, the combination of LOFU with an ablativehypofractionated Cone Beam computed tomography (CT) image-guidedradiation therapy (IGRT) results in synergistic control of primarytumors and also causes reduction in spontaneous pulmonary metastases andprolongs recurrence free survival in immunocompetent mice (see Example1). In addition, LOFU was found to sensitize cancer cells (prostatecancer in the example) to a chemotherapeutic (see Example 2).

In an exemplary embodiment, the LOFU (also termed “acoustic primingtherapy” herein) involves the application of ulstrasound at an acousticpower between 10 and 1000 W/cm² spatial peak temporal average intensity(Ispta) in a treatment zone, with the ultrasound applied continuouslyfor a time in the range of 0.5 to 5 seconds, wherein the frequency is inthe range of 0.01 to 10 MHz and the mechanical index is less than 4.Mechanical Index (MI) is the rarefaction pressure in units of MPa overthe square root of the central frequency in units of MHz. The energy andintensity of ultrasound applied is intended to fall between energies andintensities of ultrasound that either induce primarily ablative effectsor primarily diagnostic effects.

As explained in more detail below, the various treatment methodsdiscussed herein may be administered using a LOFU or acoustic primingtherapy device that includes a transducer that generates acoustic powerbetween 10 and 1000 W/cm² spatial peak temporal average intensity(I_(spta)) in a treatment zone. The ultrasound is applied continuouslyfor a time in the range of 0.5 to 5 seconds or pulsed with pulsedurations of 1 to 100 ms, wherein the frequency is in the range of 0.01to 10 MHz. In some embodiments the frequency is in the range of 0.05 to5 MHz. In some embodiments the frequency range is from 0.1 to 2 MHz. Insome embodiments the minimum diameter of any ultrasound beam in thetreatment zone is about 1 cm. In an embodiment, the LOFU is administeredat 10 to 100 W/cm² I_(spta) in the area of treatment. In an embodiment,the LOFU is administered at 100 to 200 W/cm² I_(spta) in the area oftreatment. In an embodiment, the LOFU is administered at 300 to 400W/cm² I_(spta) in the area of treatment. In an embodiment, the LOFU isadministered at 400 to 500 W/cm² I_(spta) in the area of treatment. Inan embodiment, the LOFU is administered at 500 to 600 W/cm² I_(spta) inthe area of treatment. In an embodiment, the LOFU is administered at 600to 700 W/cm² I_(spta) in the area of treatment. In an embodiment, theLOFU is administered at 700 to 800 W/cm² I_(spta) in the area oftreatment. In an embodiment, the LOFU is administered at 800 to 900W/cm² I_(spta) in the area of treatment. In an embodiment, the LOFU isadministered at 900 to 1000 W/cm² I_(spta) in the area of treatment. Inan embodiment, the ultrasound is applied for a time in the range of 0.5to 1 second. In an embodiment, the ultrasound is applied for a time inthe range of 1 to 2 seconds. In an embodiment, the ultrasound is appliedfor a time in the range of 2 to 3 seconds. In an embodiment, theultrasound is applied for a time in the range of 4 to 5 seconds. Inembodiment, the ultrasound is applied at a frequency of 0.01 to 1 MHz.In embodiment, the ultrasound is applied at a frequency of 1 to 2 MHz.In embodiment, the ultrasound is applied at a frequency of 2 to 3 MHz.In embodiment, the ultrasound is applied at a frequency of 3 to 4 MHz.In embodiment, the ultrasound is applied at a frequency of 4 to 5 MHz.In embodiment, the ultrasound is applied at a frequency of 5 to 6 MHz.In embodiment, the ultrasound is applied at a frequency of 6 to 7 MHz.In embodiment, the ultrasound is applied at a frequency of 7 to 8 MHz.In embodiment, the ultrasound is applied at a frequency of 8 to 9 MHz.In embodiment, the ultrasound is applied at a frequency of 9 to 10 MHz.

A method is provided for increasing the efficacy of a chemotherapy in asubject comprising administering to the subject (i) an amount of lowintensity focused ultrasound (LOFU) and (ii) an amount of achemotherapeutic drug, wherein the chemotherapeutic drug effectsendoplasmic reticulum (ER) stress and/or unfolded protein response (UPR)in a tumor cell, wherein the amounts (i) and (ii) together aresufficient to increase the efficacy of the chemotherapy.

Also provided is a method of increasing the efficacy of a chemotherapyin a predetermined volume of tissue in a subject which volume is lessthan the whole subject, comprising (i) administering to the subject anamount of a chemotherapeutic drug, wherein the chemotherapeutic drugeffects endoplasmic reticulum (ER) stress and/or unfolded proteinresponse (UPR) in a tumor cell and (ii) administering to thepredetermined volume of tissue in a subject an amount of low intensityfocused ultrasound (LOFU), wherein the amounts of (i) and (ii) togetherare sufficient to increase efficacy of the chemotherapy within thepredetermined volume of tissue.

Also provided is a method of treating a tumor in a subject, wherein thetumor is resistant to a chemotherapeutic drug, comprising:

receiving identification of the subject as having a tumor resistant to aspecified chemotherapeutic drug;

administering (i) an amount of low intensity focused ultrasound (LOFU)and (ii) an amount of the specified chemotherapeutic drug,

wherein the amounts (i) and (ii) together are sufficient to treat thetumor.

Also provided is a method of treating a chemoresistant tumor in asubject, wherein the tumor has become chemoresistant to a previouslyadministered chemotherapeutic drug, comprising:

administering to the subject (i) an amount of low intensity focusedultrasound (LOFU) and (ii) an amount of the chemotherapeutic drug,

wherein the amounts (i) and (ii) together are sufficient to treat thechemoresistant tumor.

In an embodiment of the methods, the chemotherapeutic drug effectsendoplasmic reticulum (ER) stress and/or unfolded protein response (UPR)in a tumor cell.

In an embodiment of the methods, the chemotherapeutic drug haspreviously been administered to the subject a plurality of times andwherein the tumor has been diagnosed as resistant to thechemotherapeutic drug subsequent to an initial administration of thechemotherapeutic drug.

In an embodiment of the methods involving chemoresistance, the methodscan further comprising receiving identification of the subject as havingthe tumor chemoresistant to a previously administered chemotherapeuticdrug.

In an embodiment of the methods, the chemotherapeutic drug effects UPRin a tumor cell.

In an embodiment of the methods, the chemotherapeutic drug effects ERstress in a tumor cell.

In an embodiment of the methods, the amounts of (i) and (ii) togetherare sufficient to induce apoptosis of tumor cells or increase apoptosisof tumor cells.

In an embodiment of the methods, the amount of administeredchemotherapeutic drug alone, in the absence of increasing the efficacy,is a sub-therapeutic dose with regard to treating a tumor.

In an embodiment of the methods, the LOFU administered is directed at alocation of the tumor in the subject.

In an embodiment of the methods, the low intensity focused ultrasound(LOFU) is administered to the subject prior to, or concurrent with, thechemotherapy or the radiotherapy or the immunotherapy. In an embodimentof the methods, the LOFU is administered to the subject prior to theradiotherapy being administered. In an embodiment of the methods, theLOFU is administered to the subject prior to the chemotherapy beingadministered. In an embodiment of the methods, the LOFU is administeredto the subject prior to the immunotherapy being administered. In anembodiment of the methods, the LOFU is administered to the subjectconcurrent with the radiotherapy being administered. In an embodiment ofthe methods, the LOFU is administered to the subject concurrent with thechemotherapy being administered. In an embodiment of the methods, theLOFU is administered to the subject concurrent with the immunotherapybeing administered.

In an embodiment of the methods, the chemotherapeutic drug is an HSP90inhibitor. In an embodiment the HSP90 inhibitor is 17AAG (tanespimycinor 17-N-allylamino-17-demethoxygeldanamycinan). In an embodiment, thechemotherapy drug is an HSP90 inhibitor. An example of an HSP90inhibitor is 17AAG (tanespimycin or17-N-allylamino-17-demethoxygeldanamycinan). In an embodiment, thechemotherapy drug is an alkylating agent. In an embodiment, thechemotherapy drug is trabectidin. In an embodiment, the chemotherapydrug is a mustard gas derivative. In an embodiment, the chemotherapydrug is a metal salt. In an embodiment, the chemotherapy drug is a plantalkaloid. In an embodiment, the chemotherapy drug is a antitumorantibiotic. In an embodiment, the chemotherapy drug is anantimetabolite. In an embodiment, the chemotherapy drug is atopoisomerase inhibitor. In an embodiment, the chemotherapy drug is aprotesomal inhibitor. In an embodiment, the chemotherapy drug is achemotherapeutic NSAID. In an embodiment, the chemotherapy drug is oneof the miscellaneous antineoplastics listed hereinbelow.

In an embodiment of the methods, the LOFU is delivered via an ultrasoundbeam from an ultrasound machine comprising a transducer and the machineand subject are positioned such that at least a portion of the tumor ispositioned at the focus of the transducer. In an embodiment of themethods, the LOFU is delivered to at least a portion of the tumor andthe position of the tumor in the subject is monitored via an imagingtechnique. In an embodiment of the methods, the imaging technique ismagnetic resonance imaging. In an embodiment of the methods, the imagingtechnique is computed tomography. In an embodiment of the methods, theimaging technique is ultrasound imaging.

In an embodiment of the methods, the LOFU is administered to multiplevolumes within the tumor at least once over a period of time of lessthan one hour.

In an embodiment of the methods, the LOFU is non-ablative. In anembodiment of the methods, the LOFU does not cause cavitation in thetissue it is administered to.

In an embodiment of the methods, an ultrasound component of the LOFU isadministered at a frequency of from 0.5 MHz to 1.5 MHz. In an embodimentof the methods, the LOFU is administered for 1 to 3 seconds. In anembodiment of the methods, the LOFU is administered by an ultrasoundbeam such that in the treatment zone in situ intensity is from 250 W/cm²to 750 W/cm² at 1 mm to 75 mm tissue depth in the subject.

In an embodiment of the methods, the LOFU is administered over theentire tumor volume. In an embodiment of the methods, the methoddelivers energy in the range of 300 to 3000 joules per cc of tumor tothe tumor. In an embodiment of the methods, high intensity focusedultrasound (HIFU) is not administered to the subject. In an embodiment,HIFU is focused ultrasound that effects a tissue temperatures in thefocal zone of about 80° C. or above. HIFU causes increase temperature upto 60 to 85° C. for few seconds of exposure time to solid tissue and/orcauses thermal ablation in the tissue. Thermal ablation is usuallyachieved with power intensity of greater than 1 kW/cm² with reportedfrequency of 0.8 to 7 MHz. On the other hand, LOFU can be achieved withpower intensity of, for example, 1 to 3 W/cm² and frequency of 0.5 to 3MHz (see other LOFU ranges herein, however). LOFU can be continuous(100% DC) or pulsed (<100% DC, some literatures referred to as lowintensity pulsed ultrasound or LIPUS) focused ultrasound by adjustingthe duty cycle. Continuous LOFU at 1 MHz and 1 W/cm² for 10 minutes canproduce a 0.1° C. elevation in tissue. In-vivo experiments on muscletissue show that sonication at 1 MHz frequency increases temperature ata rate of 0.04° C./min at 0.5 W/cm²; 0.16° C./min at 1.0 W/cm²; 0.33°C./min at 1.5 W/cm²; 0.38° C./min at 2.0 W/cm².

In an embodiment of the methods, the effect of the amount ofradiotherapy and the amount of LOFU is synergistic in treating thetumor.

In an embodiment of the methods, the subject is human.

In an embodiment of the methods, the tumor is a tumor of the prostate,breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach,esophagus, testes, ovary, uterus, endometrium, liver, small intestine,appendix, colon, rectum, bladder, gall bladder, pancreas, kidney,urinary bladder, cervix, vagina, vulva, prostate, thyroid or skin, heador neck, glioma or soft tissue sarcoma. In an embodiment of the methods,the tumor is a prostate cancer.

In an embodiment of the methods, the metastasis is a lung metastasis.

In an embodiment of the methods, the LOFU is administered with a devicecomprising:

a control system that generates a frequency waveform; and

one or more transducers configured to produce ultrasound based on afrequency waveform between 1 and 1000 W/cm² spatial peak temporalaverage acoustic output intensity (I_(spta)) in a treatment zone,wherein ultrasound is applied continuously to the treatment zone for atime in the range of from 0.5 to 5 seconds, wherein ultrasound frequencyis in the range of 0.01 to 10 MHz and wherein mechanical index of anybeam is less than 4. In an embodiment of the methods, each of the one ormore transducers are configured to produce ultrasonic beams based on thefrequency waveform with central frequencies in the range of 0.05 to 5MHz and an acoustic output intensity of between 20 and 1000 W/cm². In anembodiment of the methods, each of the one or more transducers areconfigured to produce ultrasonic beams based on the frequency waveformwith central frequencies in the range of 0.5 to 1.5 MHz and an acousticoutput intensity of between 20 and 1000 W/cm². In an embodiment of themethods, each transducer is configured to produce columnated ultrasoundsuch that the beam profile waist at −3 dB is not less than 5 mm in atreatment zone. In an embodiment of the methods, one or more beams aremechanically moved during treatment. In an embodiment of the methods,the one or more transducers comprise two or more transducers configuredto operate sequentially or simultaneously and produce ultrasound ofaverage spatial peak 250 W/cm² in a treatment zone during a treatmentperiod. In an embodiment of the methods, the one or more transducers areconfigured produce ultrasound having a frequency within the range of 10kHz to 300 kHz. In an embodiment of the methods, the one or moretransducers are configured produce ultrasound having a frequency withinthe range of 300 kHz to 3 MHz. In an embodiment of the methods, one ormore transducers operate at a frequency of 300 kHz to 3 MHz and one ormore transducers operates at a frequency of between 30 and 300 kHz. Inan embodiment of the methods, two or more ultrasound transducersgenerate ultrasound beams that pass through a treatment zone, with eachbeam having an I_(spta) in the intersection zone in the range of 10 to500 W/cm². In an embodiment of the methods, the treatment time is lessthan 5 seconds per cubic centimeter of tumor. In an embodiment of themethods, two transducers generate ultrasound beams that intersect withina treatment zone, with each beam having an I_(spta) in the intersectionzone in the range of 50 to 500 W/cm². In an embodiment of the methods,three transducers generate ultrasound beams that pass through atreatment zone, with each beam having an I_(spta) in the intersectionzone in the range of 50 to 500 W/cm². In an embodiment of the methods,the one or more transducers produce ultrasonic beams that aresubstantially in phase with one another within the treatment zone. In anembodiment of the methods, two ultrasound beams emanating from separateultrasound transducers are substantially in phase and intersect within atreatment zone, and each beam has an acoustic power spatial peakintensity in the intersection zone in the range of 70 to 100 W/cm² andthe ultrasound is applied continuously from 1 to 5 seconds.

In an embodiment of the methods, three ultrasound beams emanating fromseparate ultrasound transducers are substantially in phase and intersectwithin a treatment zone, and each beam has an acoustic power spatialpeak intensity in the intersection zone in the range of 50 to 70 W/cm²and the ultrasound is applied continuously for 1 to 5 seconds. In anembodiment of the methods, ultrasonic beams originating from separatetransducers each produce an I_(spta) in the range of approximately 100to 1000 W/cm² in the treatment zone. In an embodiment of the methods, atleast one transducer generates an ultrasonic beam with a high intensitydiameter that is substantially larger in size than the treatment zoneand is directed such that the treatment zone is entirely within thebeam. In an embodiment of the methods, an intense treatment zone isformed where two or more ultrasound beams cross paths, the intensetreatment zone being equal to or greater than about 1 cm perpendicularto the transmitted energy direction and also equal to or greater thanabout 1 cm parallel to the transmitted direction. In an embodiment ofthe methods, acoustic pressure applied to a treatment zone from eachtransducer is 0.1 to 10 MPa. In an embodiment of the methods, the numberof transducers that provide the intense ultrasound treatment zone isbetween 1 and 1000. In an embodiment of the methods, the ultrasound fromthe one or more transducers is applied continuously during the treatmenttime. In an embodiment of the methods, the ultrasound is produced with aduty cycle in the range of 1 on time units to 9 off time units. In anembodiment of the methods, the transducers are configured to produceultrasound in single frequency tones or multi-frequency chirps. In anembodiment of the methods, the one or more transducers are operatedsequentially in time. In an embodiment of the methods, the total energydelivered to the target tissue and desired margin around the targettissue for the entire course of the application is greater than that tosurrounding tissues. In an embodiment of the methods, the one or moretransducers are configured so that the frequency of ultrasound is sweptduring application. In an embodiment of the methods, the one or moretransducers comprise two-dimensional phased arrays. In an embodiment ofthe methods, the one or more transducers comprise annular arrays. In anembodiment of the methods, the one or more transducers comprisethree-dimensional phased arrays. In an embodiment of the methods, theone or more transducers are incorporated into one or more endoscopicdevices. In an embodiment of the methods, the one or more transducersare incorporated into a magnetic resonance imaging machine.

In an embodiment of the methods, the one or more transducers areincorporated into a radiotherapy treatment machine.

In an embodiment of the methods, the one or more transducers areconfigured to produce ultrasound so that the maximum temperature reachedin the treatment zone is less than 45° C. during a treatment whereultrasound is applied to the treatment zone for about 2 seconds or less.

In an embodiment of the methods, the one or more transducers areconfigured to produce ultrasound so that the maximum temperature reachedin the treatment zone is less than 50° C. during a treatment whereultrasound is applied to the treatment zone for about 2 seconds or less.

In an embodiment of the methods, the one or more transducers areconfigured to produce ultrasound so that the maximum temperature reachedin the treatment zone is less than 55° C. during a treatment whereultrasound is applied to the treatment zone for about 2 seconds or less.

In an embodiment of the methods, the LOFU and radiotherapy areadministered by a system comprising:

a LOFU device comprising:

a control system that generates a frequency waveform; and

one or more transducers configured to produce ultrasound based on afrequency waveform between 1 and 1000 W/cm² spatial peak temporalaverage acoustic output intensity (I_(spta)) in a treatment zone,wherein the ultrasound is applied continuously for a time in the rangeof from 0.5 to 5 seconds, and wherein ultrasound frequency is in therange of 0.01 to 10 MHz;a radiotherapy treatment machine; anda control system operatively configured to control the LOFU device andthe radiotherapy treatment machine so that a first amount of theultrasound and a second amount of radiotherapy are administered to asubject, wherein the first and second amounts together are sufficient totreat a tumor in the subject.

A method of treating a tumor in a subject is provided comprisingadministering to the subject (i) an amount of low intensity focusedultrasound (LOFU) and (ii) an amount of chemotherapy or an amount ofradiotherapy or an amount of immunotherapy, wherein the amounts of (i)and (ii) together are sufficient to treat a tumor.

In an embodiment of the method, the amount of LOFU and the amount ofradiotherapy are administered to the subject. In another embodiment ofthe method, the amount of LOFU and the amount of radiotherapy areadministered to the subject. In another embodiment of the method, theamount of LOFU and the amount of immunotherapy are administered to thesubject.

A method of treating a tumor in a subject is provided comprisingadministering to the subject (i) an amount of low intensity focusedultrasound (LOFU) and (ii) an amount of a targeted anti-cancer therapywherein the amounts of (i) and (ii) together are sufficient to treat atumor. In an embodiment, the targeted therapy comprises a mAb directedto Her2 or VEGFR. In an embodiment, the targeted therapy comprises atyrosine kinase inhibitor.

Also provided is a method of inhibiting metastasis of a tumor in asubject, comprising administering to a subject having a tumor an amountof low intensity focused ultrasound (LOFU) and an amount of aradiotherapy, wherein the amounts together are sufficient to inhibitmetastasis of a tumor in a subject.

In the methods, the radiotherapy can be ablative hypofractionatedradiation therapy.

Preferably, in the methods the LOFU is directed at a location of thetumor in the subject.

Also provided is a method of reducing the effective dose of ananti-cancer chemotherapy required to treat a tumor in a subjectcomprising administering to the subject undergoing the anti-cancerchemotherapy an amount of low intensity focused ultrasound (LOFU)sufficient to reduce the effective dose of the anti-cancer chemotherapyrequired to treat a tumor.

In an embodiment of each of the methods, the LOFU is administered to thesubject prior to, or concurrent with, the chemotherapy or theradiotherapy or the immunotherapy.

In an embodiment the LOFU is administered to the subject prior to theradiotherapy being administered.

In the methods wherein an anti-cancer chemotherapy is administered, inan embodiment the anti-cancer chemotherapy comprises administration ofan HSP90 inhibitor to the subject. The HSP90 inhibitor can be 17AAG(tanespimycin or 17-N-allylamino-17-demethoxygeldanamycinan). In anembodiment, the chemotherapy drug is an alkylating agent. In anembodiment, the chemotherapy drug is trabectidin. In an embodiment, thechemotherapy drug is a mustard gas derivative. In an embodiment, thechemotherapy drug is a metal salt. In an embodiment, the chemotherapydrug is a plant alkaloid. In an embodiment, the chemotherapy drug is aantitumor antibiotic. In an embodiment, the chemotherapy drug is anantimetabolite. In an embodiment, the chemotherapy drug is atopoisomerase inhibitor. In an embodiment, the chemotherapy drug is aprotesomal inhibitor. In an embodiment, the chemotherapy drug is achemotherapeutic NSAID. In an embodiment, the chemotherapy drug is oneof the miscellaneous antineoplastics listed hereinbelow.

In an embodiment of the methods, the LOFU is delivered via an ultrasoundbeam from an ultrasound machine comprising a transducer and the machineand subject are positioned such that the at least a portion of the tumoris positioned at the focal length of the transducer.

In an embodiment of the methods, the LOFU is delivered to at least aportion of the tumor and the position of the tumor is monitored via animaging technique. Magnetic resonance imaging can be such an imagingtechnique.

In the methods, the LOFU can be administered to multiple points withinthe tumor at least once over a period of time of less than one hour.

In an embodiment of the methods, the LOFU is non-ablative.

In an embodiment of the methods, the LOFU is administered at a frequencyof from 0.5 MHz to 1.5 MHz.

In an embodiment of the methods, the LOFU is administered for 1.5-3seconds

In an embodiment of the methods, the LOFU is administered by anultrasound beam such that at the focus of the ultrasound beam the insitu intensity is from 250 W/cm² to 750 W/cm². In an embodiment of themethods, the LOFU is administered by an ultrasound beam such that at thefocus of the ultrasound beam the in situ intensity is from 250 W/cm² to750 W/cm² at 1 mm to 75 mm tissue depth in the subject. In an embodimentof the methods, the LOFU is administered by an ultrasound beam such thatat the focus of the ultrasound beam the in situ intensity is from 350W/cm² to 650 W/cm² at 1 mm to 75 mm tissue depth in the subject. In anembodiment of the methods, the LOFU is administered by an ultrasoundbeam such that at the focus of the ultrasound beam the in situ intensityis from 450 W/cm² to 550 W/cm² at 1 mm to 75 mm tissue depth in thesubject.

The LOFU can be administered over the entire tumor volume, or can beadministered over a portion of the tumor volume. In a preferredembodiment the LOFU is administered over the entire tumor volume.

In an embodiment, of the method the LOFU delivers at least 500 to 5000joules of energy per cc of tumor tissue through the tumor. In anembodiment, of the method the LOFU delivers at least 1000 to 4000 joulesof energy per cc of tumor tissue. In an embodiment, of the method theLOFU delivers at least 2000 to 3000 joules of energy per cc of tumortissue through the tumor.

In an embodiment, high intensity focused ultrasound (HIFU) is notadministered to the subject. In an embodiment, high intensity focusedultrasound has not been administered to the subject. In an embodiment,high intensity focused ultrasound has not been administered to thetumor. In an embodiment where LOFU is administered to the subject beforethe anti-cancer therapy, high intensity focused ultrasound is notadministered to the subject after the LOFU is administered and beforethe anti-cancer therapy is administered.

In an embodiment, the anti-cancer therapeutic effect of the amount ofradiotherapy and the amount of LOFU is synergistic.

In an embodiment, the LOFU adminstered raises the tissue/tumortemperature to between 40° C.-45° C. In an embodiment, the LOFUadminstered raises the tissue/tumor temperature to no more than 40° C.In an embodiment, the LOFU adminstered raises the tissue/tumortemperature to no more than 45° C. In an embodiment, the LOFUadminstered raises the tissue/tumor temperature to no more than 50° C.HIFU will generally raise tissue temperatures more than this.

In an embodiment, the LOFU is administered for 0.5 to 3 seconds. In anembodiment, the LOFU is administered for 1.5 to 3 seconds. In anembodiment, the LOFU is administered with a 100% duty cycle. In anembodiment, the LOFU is administered with one of the separateembodiments of a 10, 20, 30, 40, 50, 60, 70, 80 or 90% duty cycle.

Also provided is a method of sensitizing a tumor in a subject to anamount of an anti-cancer therapy the method comprising administering tothe subject, prior to, during or after the anti-cancer therapy, anamount of low intensity focused ultrasound (LOFU) effective to sensitizea tumor in a subject to an amount of an anti-cancer therapy. In anembodiment, the anti-cancer therapy comprises a chemotherapy, or aradiotherapy, or an immunotherapy, or a targeted therapy, or a surgery.In an embodiment, the anti-cancer therapy comprises a chemotherapy. Inan embodiment, the anti-cancer therapy comprises an immunotherapy. In anembodiment, the anti-cancer therapy comprises a radiotherapy. In anembodiment, the anti-cancer therapy comprises surgery, for example, toexcise the tumor. The method can further comprise administering theanti-cancer therapy to the subject. Sensitizing a tumor to an amount ofan anti-cancer therapy makes the tumor more susceptible to thetreatment. For example, a parameter by which tumor treatment may bemeasured, such as tumor volume reduction, is greater for a given amountof an anti-cancer therapy applied to the sensitized tumor as compared tothe same amount of an anti-cancer therapy applied to a non-sensitiziedtumor of equivalent mass, vascularity, position and type in the same oran equivalent subject. In an embodiment, the amount of LOFU effective tosensitize a tumor in a subject to an amount of an anti-cancer therapyand the anti-cancer therapy are synergistic in effect.

In any of the methods described herein, the subject is a mammal. In anembodiment, the subject is a human.

The tumor referred to in the methods can be a tumor of the prostate,breast, nasopharynx, pharynx, lung, bone, brain, sialaden, stomach,esophagus, testes, ovary, uterus, endometrium, liver, small intestine,appendix, colon, rectum, bladder, gall bladder, pancreas, kidney,urinary bladder, cervix, vagina, vulva, prostate, thyroid or skin, heador neck, or is a glioma or a soft tissue sarcoma. In one embodiment, thetumor is a prostate cancer. In one embodiment, the tumor is a softtissue sarcoma. In an embodiment the primary tumor is treated. In anembodiment the secondary tumor is treated. In an embodiment, treatmentof the tumor reduces the likelihood of a secondary tumor. In oneembodiment, the metastasis comprises one or more lung metastases.

The term “tumor,” as used herein, and unless otherwise specified, refersto a neoplastic cell growth, and includes pre-cancerous and cancerouscells and tissues. Tumors usually present as a lesion or lump. In anembodiment, the tumor is a malignant neoplasm.

As used herein “metastasize” (or grammatical equivalent) means, inregard to a cancer or tumor, the spread of the cancer or tumor from oneorgan or tissue of a subject to another organ or tissue of the subjectspatially apart from the first organ or tissue.

As used herein, “treating” a tumor means that one or more symptoms ofthe disease, such as the tumor itself, vascularization of the tumor, orother parameters by which the disease is characterized, are reduced,ameliorated, inhibited, placed in a state of remission, or maintained ina state of remission. “Treating” a tumor also means that one or morehallmarks of the tumor may be eliminated, reduced or prevented by thetreatment. Non-limiting examples of such hallmarks include uncontrolleddegradation of the basement membrane and proximal extracellular matrix,migration, division, and organization of the endothelial cells into newfunctioning capillaries, and the persistence of such functioningcapillaries. In an embodiment, treating the tumor means reducing thesize or volume of the tumor.

As used herein, “inhibiting metastasis” of a tumor in a subject meansthat one or more symptoms or one or more other parameters by which thedisease is characterized, are reduced, ameliorated, or inhibited.Non-limiting examples of such parameters include uncontrolleddegradation of the basement membrane and proximal extracellular matrix,and travel of tumor cells through the bloodstream or lymphatics,invasion, dysregulated adhesion, and proliferation at secondary site,either distal or local. In an embodiment, treating the metastasis meansreducing the development or inhibiting the development of metastases.

Radiotherapy is well-known in the art. Radiotherapy as encompassedherein includes medically therapeutic radiation delivered by a machineoutside the body (external-beam radiation therapy), or from radioactivematerial placed in the body near cancer cells (internal radiationtherapy, also called brachytherapy) or systemic radiation therapy.Radiotherapy as encompassed herein includes 3-dimensional conformalradiation therapy (3D-CRT), intensity-modulated radiation therapy(IMRT), image-guided radiation therapy (IGRT), and tomotherapy. Theradiotherapy may also be part of a stereotactic radiosurgery orstereotactic body radiation therapy (SBRT). Delivery by any particlebeam known in the art is encompassed also, for example proton therapy,carbon ion therapy, or other charged particle beams depending on tumortype and location.

In an embodiment, the radiotherapy is CT image guided. In an embodiment,the radiotherapy is hypofractionated cone beam radiotherapy. In anembodiment, the radiotherapy is hypofractionated cone beam CT imageguided radiotherapy. All combinations of the various elements describedherein are within the scope of the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

Chemotherapeutic drugs which may be used in the invention are various.Examples of chemotherapeutic drugs include:

alkylating agents (e.g. trabectidin((1′R,6R,6aR,7R,13S,14S,16R)-6′,8,14-trihydroxy-7′,9-dimethoxy-4,10,23-trimethyl-19-oxo-3′,4′,6,7,12,13,14,16-octahydrospiro[6,16-(epithiopropano-oxymethano)-7,13-imino-6aH-1,3-dioxolo[7,8]isoquino[3,2-b][3]benzazocine-20,1′(2′H)-isoquinolin]-5-ylacetate));mustard gas derivatives (e.g. mechlorethamine, cyclophosphamide,chlorambucil, melphalan, and ifosfamide);ethylenimines (e.g. thiotepa and hexamethylmelamine);alkylsulfonates (e.g. busulfan);hydrazines and triazines (e.g. altretamine, procarbazine, dacarbazineand temozolomide); nitrosureas (e.g. carmustine, lomustine andstreptozocin);metal salts (e.g. carboplatin, cisplatin, and oxaliplatin);plant alkaloids (e.g. vinca alkaloids such as vincristine, vinblastineand vinorelbine, taxanes such as paclitaxel and docetaxel,podophyllotoxins such as etoposide and tenisopide, camptothecan analogs(topoisomerase inhibitors) such as irinotecan and topotecan);antitumor antibiotics (e.g. anthracyclines: doxorubicin, daunorubicin,epirubicin, mitoxantrone, and idarubicin; chromomycins: dactinomycin andplicamycin; miscellaneous ones such as mitomycin and bleomycin);antimetabolites (e.g. folic acid antagonists: methotrexate; pyrimidineantagonists: 5-fluorouracil, foxuridine, cytarabine, capecitabine, andgemcitabine; purine antagonist: 6-mercaptopurine and 6-thioguanine;adenosine deaminase inhibitors: cladribine, fludarabine, nelarabine andpentostatin);topoisomerase inhibitors (e.g. ironotecan, topotecan; amsacrine,etoposide, etoposide phosphate, teniposide);protesomal inhibitors;Chemotherapeutic NSAIDS; andmiscellaneous antineoplastics (e.g. ribonucleotide reductase inhibitor:hydroxyurea; adrenocortical steroid inhibitor: mitotane; enzymes:asparaginase and pegaspargase; antimicrotubule agent: estramustine;retinoids: bexarotene, isotretinoin, tretinoin (ATRA).

Chemotherapeutic drugs which effect endoplasmic reticulum (ER) stressand/or effect unfolded protein response (UPR) in cells are known in theart. For example, protesomal inhibitors such as e.g. Bortezomib(Velcade; previously known as PS-341), elicit an ER stress response.Also, protesomal inhibitors such as e.g. Bortezomib elicit the UPR.Histone deacetylase (HDAC) inhibitors elicit an ER stress response.Chemotherapeutic NSAIDS (e.g. indomethacin, diclofenac, and celecoxib)can elicit an ER stress response and can elicit the UPR. Estrogenreceptor a inhibitors such BHPI (1,3-dihydro-3,3-bis(4-hydroxyphenyl)-7-methly-2H-indol-2-one) can activatethe UPR. Platinum-containing anti-cancer drugs: cisplatin is known toelicit an ER stress response. The taxane family of drugs: paclitaxel isknown to elicit an ER stress response. Anthracycines such as doxorubicinare known to elicit ER stress. Cyclophosphamide is known to elicit ERstress. See also Table 2 of Hetz et al., Nature Reviews, Drug Discovery,12:703-719 (September, 2013), hereby incorporated by reference.

The endoplasmic reticulum (ER) is the site of synthesis and folding ofsecreted, membrane-bound and some organelle-targeted proteins. The ER ishighly sensitive to stresses that perturb cellular energy levels, theredox state or Ca²⁺ concentration. Such stresses reduce theprotein-folding capacity of the ER, which can result in the accumulationand aggregation of unfolded proteins and/or an imbalance between theload of resident and transit proteins in the ER and the organelle'sability to process that load. This condition is referred to herein, andin the art, as “ER stress”. The ER stress response can promote cellularrepair and sustained survival by reducing the load of unfolded proteinsthrough global attenuation of protein synthesis and/or upregulation ofchaperones, enzymes and structural components of the ER, which enhanceprotein folding. This response is collectively termed as the unfoldedprotein response (UPR). Accumulation of unfolded proteins causesdissociation of GRP78 from PERK, ATF6 and IRE1, thereby initiating theUPR.

TABLE 1 Non-limiting examples of chemotherapies that can be used withLOFU Selected interactions Types of action Drug name Mechanism of Actionwith LOFU Block proteosome/ Decrease the degradation of degradation ofmisfolded misfolded protein protein that LOFU induces thereby increasingthe amount of unfolded protein response to tip towards apoptosis.Bortezomib proteosome inhibitor ATF-4 induction by UPR can conferresistance to Bortezomib 3-methyladenine an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase) prevented induction of ATG5 andactivation of LC3-II and blocked autophagosome formation polyphenol(green tea) proteosome inhibitor epigallocatechin gallate genisteinProteosome inhibitor curcumin Proteosome inhibitor resveratrolProteosome inhibitor/ represses XBP-1 prosurvival signaling Inhibitor ofDuring times of stress, autophagy the cells will induce autophagy toimmediately replenish depleting building blocks. Inhibiting the processof autophagy in cells treated with LOFU is expected to result in cellsthat are not able to immediately respond to the stress and therebyinducing apoptosis. 15,16- induce UPR via Dihydrotanshinone I proteosomeinhibition (Tanshen root) Chloroquine inhibitor of autophagy Inducer ofDecreasing the unfolded autophagy protein response sparked by LOFU andmaking cells less sensitive. If autophagy is activated for long enoughthen can push cells into pro- apoptotic state. A combination of increasemacroautophagy plus increased UPR can provide an immunological response.rapamycin mTOR inhibitor temsirolimus mTOR inhibitor 4-O-carboxymethylagonist of the nuclear ascochlorin hormone receptor PPARγ; ER stress-induced autophagy and apoptosis ER stress inducer Combining twodifferent sources of UPR inducing agents can provide an additive effectto cancer killing. Celecoxib Cox-2 inhibitor - by causing leakage ofcalcium from ER into cytosol Verapamil Ca channel inhibitor Enhancing ERstress signaling Ritonavir inhibits protein degradation that'ssynergistic with proteosome inhibitor 3-thia fatty acid Inducer of ERstress tetradecylthioacetic acid Nelfinavir modulates CHOP expressionDamage DNA Inhibiting the repair structure/prevents mechanism to preventDNA synthesis cellular repair after LOFU damage. cisplatin induce ERstress and dmg DNA gemcitabine Nucleoside analog Block protein synthesissalubrinal inhibitor of eIF2a phosphotase Cycloheximide Increase deathsignaling TRAIL chemical chaperones 4-phenylbutyric acid chemicalchaperone which rescue the mutant alpha1- antitrypsin phenotypechaperones inhibitor geldanamycin HSP90 inhibitor 17-allyamino-17- HSP90inhibitor demethoxygeldanamycin (17AAG) 17-dimethylamino- HSP90inhibitor ethylamino-17- demethoxygeldanamycin (17DMAG) blockingsonoporation repair vacuolin a newly discovered small organic moleculethat blocks the wounding- and Ca2+- triggered fusion of lysosomes withthe plasma membrane

In an embodiment of the methods, the chemotherapy drug is an HSP90inhibitor. An example of an HSP90 inhibitor is 17AAG (tanespimycin or17-N-allylamino-17-demethoxygeldanamycinan). In an embodiment, thechemotherapy drug is an alkylating agent. In an embodiment, thechemotherapy drug is trabectidin. In an embodiment, the chemotherapydrug is a mustard gas derivative. In an embodiment, the chemotherapydrug is a metal salt. In an embodiment, the chemotherapy drug is a plantalkaloid. In an embodiment, the chemotherapy drug is an antitumorantibiotic. In an embodiment, the chemotherapy drug is anantimetabolite. In an embodiment, the chemotherapy drug is atopoisomerase inhibitor. In an embodiment, the chemotherapy drug is aprotesomal inhibitor. In an embodiment, the chemotherapy drug is achemotherapeutic NSAID. In an embodiment, the chemotherapy drug is oneof the miscellaneous antineoplastics listed hereinabove.

Other non-limiting examples of chemotherapy drugs or agents encompassedby the invention, unless otherwise stated, include anthracyclines,maytansinoids, alkylating agents, anti-metabolites, plant alkaloids orterpenoids, and cytotoxic antibiotics. In embodiments, the chemotherapyagent is cyclophosphamide, bleomycin, etoposide, platinum agent(cisplatin), fluorouracil, vincristine, methotrexate, taxol, epirubicin,leucovorin (folinic acid), or irinotecan.

Anti-tumor immunotherapies encompassed herein (i) include monoclonalantibodies (including naked, chemo-, radio- or toxin-conjugatedantibodies and also bispecific antibodies), relevant antigen-bindingfragments thereof such as fragments comprised of Fab or scFv fragments,that that bind with high affinity to cancer-associated biomoleculartargets; (ii) those that are non-specific with respect to tumor cellsand tumor cell antigens; anti-tumor immunotherapies e.g. cytokines,interleukins, interferons, GM-CSF, small organic molecules (e.g. 1,500daltons or less) or other drugs that bind to cytokines or cytokinereceptors; and materials that target checkpoints including but notlimited to one of CTLA-4, PD-1, PDL-1, and other small organicmolecules, peptides and aptamers that target immune responses. In anembodiment, immunotherapy as used herein excludes bacteria-basedanticancer or anti-tumor immunotherapies. In one embodiment,immunotherapy as used herein excludes Listeria-based immunotherapies.

In an embodiment, increasing efficacy of a treatment means an increasein the extent of therapeutic effect achieved versus the extent achievedfor the same amount of treatment (e.g. a given treatment dose) in theabsence of the efficacy-increasing method being applied.

Also provided is an acoustic priming therapy device comprising:

a control system that generates a frequency waveform; and

one or more transducers configured to produce ultrasound based on afrequency waveform between 1 and 1000 W/cm² spatial peak temporalaverage acoustic output intensity (I_(spta)) in a treatment zone,wherein ultrasound is applied continuously to the treatment zone for atime in the range of from 0.5 to 5 seconds, wherein ultrasound frequencyis in the range of 0.01 to 10 MHz and wherein mechanical index of anybeam is less than 4.

In an embodiment of the device, each of the one or more transducers areconfigured to produce ultrasonic beams based on the frequency waveformwith central frequencies in the range of 0.05 to 5 MHz and an acousticoutput intensity of between 20 and 1000 W/cm².

In an embodiment of the device, each of the one or more transducers areconfigured to produce ultrasonic beams based on the frequency waveformwith central frequencies in the range of 0.5 to 1.5 MHz and an acousticoutput intensity of between 20 and 1000 W/cm².

In an embodiment of the device, each transducer is configured to producecolumnated ultrasound such that the beam profile waist at −3 dB is notless than 5 mm in a treatment zone.

In an embodiment of the device, one or more beams are mechanically movedduring treatment.

In an embodiment of the device, the one or more transducers comprise twoor more transducers configured to operate sequentially or simultaneouslyand produce ultrasound of average spatial peak 250 W/cm² in a treatmentzone during a treatment period.

In an embodiment of the device, the one or more transducers areconfigured produce ultrasound having a frequency within the range of 10kHz to 300 kHz.

In an embodiment of the device, the one or more transducers areconfigured produce ultrasound having a frequency within the range of 300kHz to 3 MHz.

In an embodiment of the device, one or more transducers operate at afrequency of 300 kHz to 3 MHz and one or more transducers operates at afrequency of between 30 and 300 kHz

In an embodiment of the device, two or more ultrasound transducersgenerate ultrasound beams that pass through a treatment zone, with eachbeam having an I_(spta) in the intersection zone in the range of 10 to500 W/cm²

In an embodiment of the device, the treatment time is less than 5seconds per cubic centimeter of tumor.

In an embodiment of the device, two transducers generate ultrasoundbeams that intersect within a treatment zone, with each beam having anI_(spta) in the intersection zone in the range of 50 to 500 W/cm².

In an embodiment of the device, three transducers generate ultrasoundbeams that pass through a treatment zone, with each beam having anI_(spta) in the intersection zone in the range of 50 to 500 W/cm².

In an embodiment of the device, the one or more transducers produceultrasonic beams that are substantially in phase with one another withinthe treatment zone.

In an embodiment of the device, two ultrasound beams emanating fromseparate ultrasound transducers are substantially in phase and intersectwithin a treatment zone, and each beam has an acoustic power spatialpeak intensity in the intersection zone in the range of 70 to 100 W/cm²and the ultrasound is applied continuously from 1 to 5 seconds.

In an embodiment of the device, three ultrasound beams emanating fromseparate ultrasound transducers are substantially in phase and intersectwithin a treatment zone, and each beam has an acoustic power spatialpeak intensity in the intersection zone in the range of 50 to 70 W/cm²and the ultrasound is applied continuously for 1 to 5 seconds.

In an embodiment of the device, ultrasonic beams originating fromseparate transducers each produce an I_(spta) in the range ofapproximately 100 to 1000 W/cm² in the treatment zone.

In an embodiment of the device, at least one transducer generates anultrasonic beam with a high intensity diameter that is substantiallylarger in size than the treatment zone and is directed such that thetreatment zone is entirely within the beam.

In an embodiment of the device, an intense treatment zone is formedwhere two or more ultrasound beams cross paths, the intense treatmentzone being equal to or greater than about 1 cm perpendicular to thetransmitted energy direction and also equal to or greater than about 1cm parallel to the transmitted direction.

In an embodiment of the device, acoustic pressure applied to a treatmentzone from each transducer is 0.1 to 10 MPa.

In an embodiment of the device, the number of transducers that providethe intense ultrasound treatment zone is between 1 and 1000.

In an embodiment of the device, the ultrasound from the one or moretransducers is applied continuously during the treatment time.

In an embodiment of the device, the ultrasound is produced with a dutycycle in the range of 1 on time units to 0 to 9 off time units.

In an embodiment of the device, the transducers are configured toproduce ultrasound in single frequency tones or multi-frequency chirps.

In an embodiment of the device, the one or more transducers are operatedsequentially in time.

In an embodiment of the device, the total energy delivered to the targettissue and desired margin around the target tissue for the entire courseof the application is greater than that to surrounding tissues. In anembodiment of the device, the one or more transducers are configured sothat the frequency of ultrasound is swept during application. In anembodiment of the device, the one or more transducers comprisetwo-dimensional phased arrays. In an embodiment of the device, the oneor more transducers comprise annular arrays. In an embodiment of thedevice, the one or more transducers comprise three-dimensional phasedarrays. In an embodiment of the device, the one or more transducers areincorporated into one or more endoscopic devices. In an embodiment ofthe device, the one or more transducers are incorporated into a magneticresonance imaging machine. In an embodiment of the device, the one ormore transducers are incorporated into a radiotherapy treatment machine.

In an embodiment of the device, the one or more transducers areconfigured to produce ultrasound so that the maximum temperature reachedin the treatment zone is less than 45° C. during a treatment whereultrasound is applied to the treatment zone for about 2 seconds or less.In an embodiment of the device, the one or more transducers areconfigured to produce ultrasound so that the maximum temperature reachedin the treatment zone is less than 50° C. during a treatment whereultrasound is applied to the treatment zone for about 2 seconds or less.

In an embodiment of the device, the one or more transducers areconfigured to produce ultrasound so that the maximum temperature reachedin the treatment zone is less than 55° C. during a treatment whereultrasound is applied to the treatment zone for about 2 seconds or less.

Also provided is a system comprising:

an acoustic priming therapy device comprising:

a control system that generates a frequency waveform; and

one or more transducers configured to produce ultrasound based on afrequency waveform between 1 and 1000 W/cm² spatial peak temporalaverage acoustic output intensity (I_(spta)) in a treatment zone,wherein the ultrasound is applied continuously for a time in the rangeof from 0.5 to 5 seconds, and wherein ultrasound frequency is in therange of 0.01 to 10 MHz;a radiotherapy treatment machine; anda control system operatively configured to control the acoustic primingtherapy device and the radiotherapy treatment machine so that a firstamount of the ultrasound and a second amount of radiotherapy areadministered to a subject, wherein the first and second amounts togetherare sufficient to treat a tumor in the subject.

Also provided is a system comprising:

an acoustic priming therapy device comprising:

a control system that generates a frequency waveform; and

one or more transducers configured to produce ultrasound based on afrequency waveform between 1 and 1000 W/cm² spatial peak temporalaverage acoustic output intensity (Ispta) in a treatment zone, whereinthe ultrasound is applied continuously for a time in the range of from0.5 to 5 seconds, and wherein ultrasound frequency is in the range of0.01 to 10 MHz;the acoustic priming therapy device for use in combination withchemotherapy so that a first amount of the ultrasound and a secondamount of the chemotherapy are administered to a subject, wherein thefirst and second amounts together are sufficient to treat a tumor in thesubject.

Also provided is a system comprising:

an acoustic priming therapy device comprising:

a control system that generates a frequency waveform; and

one or more transducers configured to produce ultrasound based on afrequency waveform between 1 and 1000 W/cm² spatial peak temporalaverage acoustic output intensity (Ispta) in a treatment zone, whereinthe ultrasound is applied continuously for a time in the range of from0.5 to 5 seconds, and wherein ultrasound frequency is in the range of0.01 to 10 MHz;the acoustic priming therapy device for use in combination withimmunotherapy so that a first amount of the ultrasound and a secondamount of the immunotherapy are administered to a subject, wherein thefirst and second amounts together are sufficient to treat a tumor in thesubject.

This invention will be better understood from the examples follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims that follow thereafter.

Example 1

B16 melanoma tumors suppress IL-2 and IFNγ production by tumor-specificCD4+ T cells: To determine how melanoma cells may modulate tumor inducedeffector CD4+ T cell responses, three different mouse models were used.First, B16-F1 melanoma tumors were induced in C57Bl/6J mice bysubcutaneous injection of B16 cells in the lumbar flanks. Tumors wereallowed to grow to a size of 7-8 mm and CD4+ T cells were then isolatedfrom both the ipsilateral inguinal draining lymph nodes (DLN) anddistal-contralateral non-draining cervical lymph nodes (NDLN). T cellswere also obtained from control mice that did not harbor any tumors.Supporting previous reports of tumor-antigen specific T cell tolerancein murine melanoma (18, 21), CD4+ T cells isolated from the tumor DLNproduced significantly less IL-2 than cells isolated from the distalcontralateral NDLN of the same mice, or from lymph nodes of controltumor-free mice, when stimulated ex vivo with anti-CD3 and anti-CD28antibodies. A similar but less pronounced effect was also observed forIFNγ (FIGS. 1A and B).

To confirm these data, a B16-F1 melanoma cell line that had been stablytransfected to express OVA as a surrogate tumor antigen was used. Thesecells were subcutaneously injected into OT-II mice, a mouse strain withT cells expressing a transgenic MHC class II-restricted TCR thatrecognizes the OVA323-339 peptide. T cells were collected from thesemice as described, and stimulated ex vivo using splenocytes loaded withOVA323-339 peptide. CD4+ T cells from the ipsilateral DLN again producedsignificantly reduced amounts of IL-2 and IFNγ compared to cells fromthe contralateral NDLN or from tumor free mice (FIGS. 1C and 1D).

These results were further corroborated in a third model using Tyrp1mice, which are deficient in tyrosinase-related protein 1 and bear Tcells expressing a MHC class II-restricted TCR specific for theTRP-1113-127 peptide of this endogenous melanocyte differentiationantigen. Those mice were injected with B16-F1 cells. As in the previoustwo models, IL-2 and IFNγ production by CD4+ T cells harvested from theipsilateral DLN was significantly reduced compared to cells harvestedfrom contralateral NDLN or from tumor-free mice. (FIGS. 1E and 1F).Altogether, these results support that melanoma tumors inducehyporesponsiveness in tumor antigen-specific CD4+ T cells, whichtranslates in a reduced capacity to produce effector cytokines uponre-stimulation.

Treatment of primary B16 melanoma with LOFU overcomes tumor-inducedtolerance in CD4+ T cells: HIFU is currently being used to predominantlycause tumor ablation through the generation of high amounts of heatinside the tumor tissue leading to coagulative necrosis. Although HIFUis a very effective, noninvasive ablative procedure to achieve localtumor control, it destroys the vasculature and tissue infrastructurealmost instantaneously, thereby limiting the infiltration of dendriticcells and immune cells for antigen presentation and recognition. It wasexplored whether administering LOFU would induce a non-lethalthermal/mechanical stress in the tumor tissue that could generate noveltumor antigens and/or induce the expression of stress-induced proteins,which could increase the immunogenicity of the tumor and overcometumor-induced tolerance of CD4+ T cells. In order to examine thispossibility, primary B16-F1 melanoma tumors grown on separate groups ofC57Bl/6J mice were either left untreated or treated with LOFU. Thirtysix hours after LOFU treatment, DLN and NDLN resident CD4+ T cells wereisolated from both groups of mice, and re-stimulated ex-vivo withantiCD3 and antiCD28 antibodies. CD4+ T cells from the DLN of theLOFU-treated mice produced significantly more IL-2 compared to the cellsobtained from the group of mice bearing untreated tumors. In contrast, Tcells from the corresponding NDLN produced comparable amounts of IL-2 intreated and untreated mice (FIG. 2A). A similar, but less pronouncedeffect, was observed on IFNγ production in these same experimentalgroups of mice (FIG. 2B). Overall, these results indicated that LOFUtreatments of B16 melanoma tumors appear to bolster CD4+ T cells toovercome the hyporesponsive state induced by the melanoma tumormicroenvironment, suggesting improved activation and reducedtumor-induced T cell tolerance.

It has previously been shown that melanoma tumors can induce anNFAT1-dependent program of gene expression that produces a set ofproteins which interfere with TCR signaling and directly inhibitexpression of cytokines, resulting in the establishment of functionalanergy in CD4+ T cells (21). To determine the possibility that LOFUtreatment could inhibit tumor-induced T cell tolerance by preventinganergy induction and be responsible for the increased cytokineexpression observed in the DLN resident CD4+ T cells following treatmentwith LOFU, the expression of those anergy-associated genes in CD4+ Tcells isolated from the DLN of mice bearing B16 tumors was firstmonitored and compared with the expression of those genes in T cellsharvested from NDLN of the same mice. T cells from the DLN oftumor-bearing mice expressed higher levels of anergy-associated genes,including the E3 ubiquitin ligases Grail, Cbl-b and Itch and thetranscription factor Egr2 (FIG. 2C). However, no difference in theexpression of Foxp3 was observed between the DLN and NDLN T cells intumor bearing mice, suggesting that an increased presence of regulatoryT cells was not likely contributing to the decreased CD4+ T cellresponses under the conditions used in this study (FIG. 2C).

It was then determined if treatment of B16 melanomas with LOFU wouldhave an effect on the expression of those anergy-associated genes in Tcells. To assess responses induced by endogenous tumor antigens, B16tumors growing on Tyrp1 mice were either left untreated or treated withLOFU. CD4+ T cells were isolated from the DLN and NDLN and theexpression of several anergy-associated genes was assessed. T cellsderived from the DLN showed varying degrees of upregulation of 6 of the7 anergy genes analyzed, including Grail, Itch, and Cblb, as well as thetranscription factors Egr2 and Grg4, and the protease Caspase3 (FIG.2D). Another transcription factor, Ikaros, which is also upregulated inseveral in vitro and in vivo T cell anergy models, was not significantlyupregulated in this melanoma model of tumor-induced anergy, and itslevels remained largely similar in both the DLN and NDLN derived T cells(FIG. 2D). Interestingly, when the tumors were treated with LOFU, theexpression of 5 of those genes, Grail, Itch, Cblb, Egr2 and Grg4 in theT cells isolated from the DLN was not upregulated and showed levelscomparable to the expression of these genes in the NDLN (FIG. 2D),supporting that LOFU treatment inhibited the induction of the expressionof anergy-inducing genes in tumor antigen-specific CD4+ T cells.

LOFU treated melanoma tumors are able to reactivate anergic tumorantigen-specific T cells: The results supported that tumor-induced Tcell tolerance could be overcome following LOFU treatment. Thisobservation was substantiated by the fact that the expression of severalanergy-associated genes was decreased in T cells from tumor DLNfollowing LOFU treatment of the tumor site, while activation-inducedcytokine expression was restored to levels close to those detected in Tcells isolated from distal NDLN or in T cells isolated from controlnon-tumor bearing mice.

Whether LOFU might not only prevent the induction of tumor-antigenspecific T cell anergy but also reverse established anergy and generatea productive effector response in previously tolerized T cells wasinvestigated. Naïve CD4+ T cells were isolated from spleen and lymphnodes of Tyrp1 mice, in vitro differentiated into TH1 cells andanergized by activating them through partial stimulation using withanti-CD3 antibodies in the absence of co-stimulation. As expected, Tcells became hyporesponsive and showed a profound decrease in IL-2production upon re-stimulation with anti-CD3 and anti-CD28 antibodies(FIG. 3A). These anergic cells were then re-activated with CD11c+dendritic cells loaded with lysates derived from either untreated orLOFU treated melanoma tumors. As expected, anergic Tyrp1 T cellsstimulated with dendritic cells loaded with tumor lysates from untreatedB16-F1 melanoma produced negligible amounts of IL-2. However, whendendritic cells were loaded with tumor lysates prepared fromLOFU-treated tumors, previously anergized T cells produced significantlymore IL-2 than those activated with untreated lysates (FIG. 3B). Theseresults indicate that LOFU treatment of melanoma tumors might result inthe generation of immunogenic molecules that can enable dendritic cellsto deliver activating signals that can breach tolerance, enablingotherwise anergic T cells to respond to antigen re-encounter andgenerate a productive response.

Treatment of melanoma with LOFU induces changes in the expression andsubcellular distribution of the molecular chaperones calreticulin andHsp70 in melanoma tumor cells: Activation of melanoma-specific T cellsby dendritic cells is a crucial event in determining their fate. Asuccessful antigen presentation event that is able to elicit an effectorT cell response is critically dependent on the activation state ofdendritic cells that would otherwise deliver tolerogenic stimuli. Theresults indicate that treatment of melanoma tumors with LOFU resulted inincreased CD4+ T cell activation as consequence of hinderingtumor-induced T cell tolerance. This could potentially result from thegeneration of more immunogenic dendritic cell populations. To test theeffect of LOFU on the ability of dendritic cells of efficiently presentantigens to T cells, total cells from the DLNs of tumor-bearing micewere first isolated, untreated or treated with LOFU, and immunostainedto measure the expressions of B7.1, B7.2, and MHCII on CD11c+ dendriticcell populations by flow cytometry. No significant enhancement of theexpression of these proteins in the LOFU-treated mice was detected (FIG.4A).

Trafficking of tumor antigens by molecular chaperones, includingcalreticulin and Hsp70, is also crucial for the subsequent productivepresentation of antigens to T cells (32-35). Both in vivo and in vitroapproaches were employed to detect membrane calreticulin and Hsp70 inuntreated and LOFU treated B16 melanoma tumors. Tumors, either leftuntreated or treated with LOFU, were harvested from tumor bearing mice,made into single cell suspensions and stained with a live/dead marker toassess cell viability. No differences in cell viability were observed inresponse to LOFU treatment, supporting the notion that the low energyform of FUS was not directly inducing tumor cell death (FIG. 4B). B16melanoma tumors were left untreated or exposed to LOFU treatment andtumor tissue sections were put on slides. Slides were subjected tostaining with anti-Hsp70 or anti-calreticulin antibodies for asubsequent detection by immunofluorescence. Immunofluorescence analysesof LOFU treated melanomas confirmed that LOFU induced increasedexpression of Hsp70 (FIG. 4C). Interestingly, compared to untreatedcells, LOFU treated cells also showed a change in the distribution ofcalreticulin, which appeared to accumulate in discrete regions of theplasma membrane on B16 cells (FIG. 4C). To determine if the increasedHsp70 expression also correlated with increased presence in the membraneof this protein, non-permeabilized CD45-TRP-1+B16 melanoma cells werestained for Hsp70 and cell surface expression following LOFU treatmentassessed by FACS. This analysis confirmed that LOFU treatment of B16melanomas caused increased membrane presence of Hsp-70 in tumor cells(FIG. 4D).

LOFU treatment of melanoma tumors potentiates dendritic cell-mediatedtumor antigen presentation to elicit a stronger CD4+ T cell response: Todetermine the possibility that LOFU treatment of tumors could result inenhanced stimulatory capacity of resident dendritic cells, it wasdirectly tested if lysates prepared from LOFU treated tumors couldelicit enhanced priming of antigen specific T cells leading to a morerobust effector response. For this experiment, B16-F1-OVA melanoma cellswere used to induce tumors in C57BL/6 mice. Lysates were prepared fromuntreated and LOFU treated tumors. Splenic dendritic cells and respondernaïve CD4+ T cells were isolated from C57BL/6 and OT-II tumor-free mice,respectively, and were co-cultured in the presence or absence of thedifferent tumor lysates described above. Though the OVA containing tumorlysates could act as a source of tumor antigen to prime responder Tcells, exogenous OVA323-339 peptide was also added to ensure uniformloading of dendritic cells with this peptide in all conditions and moreaccurately determine the tolerogenic or activating nature of thedifferent tumor lysates.

As expected, control responder OT-II T cells, upon activation withdendritic cells loaded with OVA323-339 peptide, showed a strong responsewith elevated levels of IL-2 production. However, lysates obtained fromuntreated tumors markedly inhibited OT-II responses and resulted in aprofound decrease in IL-2 production, even though exogenous OVA323-339peptide was added to the culture (FIG. 5A). Interestingly, as opposed tountreated lysates, lysates derived from LOFU treated tumors did not onlyhave no negative effect on the responses of OT-II cells to OVA323-339but were also able to elicit a strong activation of OT-II responder Tcells even in the absence of exogenous peptide (FIG. 5A). These resultsextended further support the observation that LOFU treatment of B16melanoma tumors prevents the negative effect on the T cell primingcapacity of dendritic cells that normally occurs in the tumormicroenvironment.

Next it was determined whether tumor DLN resident antigen presentingcells would be functionally more efficient at activating target T cellsfollowing LOFU treatment of melanoma tumors. To that effect, B16-F1melanomas were induced on C57BL/6 mice and were either left untreated ortreated with LOFU. DLN cell suspensions were depleted of T cells andused to test the capacity and DLN antigen presenting cells to activatetumor antigen specific T cells. T-cell depleted DLN cells were thusco-cultured for 24 hours with naïve Tyrp1 CD4+ T cells and lysatesprepared from B16 in vitro cultures. IL-2 production was measured byELISA to monitor responder T cell priming. Cells isolated from the DLNof LOFU treated tumor-bearing mice showed a significantly increasedability to activate Tyrp1 CD4+ T cells compared with cells isolated fromuntreated mice. (FIG. 5B). These data support that LOFU treatment of B16melanoma results in the generation of antigen presenting cells that arefunctionally more efficient at activating tumor-antigen responder Tcells.

LOFU followed by ablation of tumor by hypofractionated IGRT results inenhanced T-cell mediated control of primary melanoma lesions: In orderto further determine the consequences of our observation that LOFUtherapy can modulate tumor immunogenicity and enhance anti-tumor immuneresponses, a series of in vivo treatment strategies evaluating primarytumor control were performed using a combination of LOFU with or withouttumor ablation using daily 10 Gy hypofractionated IGRT to a total doseof 30 Gy per mouse with established B16-M1 tumors located subcutaneouslyin the right dorsal hindlimb. Treatment was initiated for all mice whentumor volume reached ˜50 mm³. Tumor volumes in each group were thenmeasured three times a week for up to 62 days (FIG. 6A). UntreatedC57BL/6 or mice treated with LOFU alone continued to experience rapidprimary tumor growth, reaching a volume of ≥300 mm³ within 10 days oftreatment, at which point a below-the-knee amputation (BKA) wasperformed (FIG. 6A). In contrast, mice within the hypofractionated IGRTor LOFU+IGRT groups experienced significant growth delay for up to3-weeks following treatment after which mice treated with IGRT alonebegan to exhibit primary tumor regrowth, reaching a volume ≥300 mm³ atapproximately 5-weeks. Remarkably, the mice in the LOFU+IGRT group had asustained response, with limited tumor growth for more than 6-weeksfollowing treatment. The reduction in tumor volume in the groupreceiving LOFU+IGRT or IGRT when compared with the untreated or LOFUalone groups was statistically significant by day 25 (P<0.05). Moreover,the reduction in tumor volume in the LOFU+IGRT group compared with IGRTalone was statistically significant by day 35 and remained statisticallysignificant (P<0.05) for the duration of the experiment. (FIG. 6A). Inaddition, mice in the LOFU+IGRT group demonstrated regression of tumorsfrom their baseline measurements and a complete tumor-free response wasseen in 4 out of 5 mice.

To corroborate the immunomodulatory effect of LOFU, similar experimentswere performed using the immunocompromised BALB/c nude model. In thesemice B16-M1 tumors grew much more rapidly, reaching ≥300 mm3approximately 1-week earlier than C57BL/6 mice. The overall treatmentresponse was similar, with untreated and LOFU alone resulting in nosignificant primary tumor control, while IGRT and LOFU+IGRT delayedprimary tumor growth (FIG. 6B). However, in both the IGRT and LOFU+IGRTtreatments, primary control was short lived. In fact, BKA was requiredin the IGRT group less than 2-weeks after starting treatment and in lessthan 3-weeks in the LOFU+IGRT group. Additionally, LOFU+IGRT inimmunocompromised mice failed to result in statistically significantprimary tumor control when compared to IGRT alone (FIG. 6B).

LOFU followed by hypofractionated IGRT results in prolonged recurrencefree survival and reduced pulmonary metastasis: Based on the data, itwas hypothesized that LOFU-induced enhanced anti-tumor T cell responsesmight augment therapeutic IGRT not only achieving better control oflocal disease, but also of microscopic disease and distant metastases.As B16-F10 is an aggressive cell line that rapidly grows to anunacceptable size if not treated, mice with primary tumors >300 mm³required BKA. Of note, by the time a BKA was performed, cells from theprimary tumor had already spread to the draining popliteal LN (data notshown). Within the subsequent weeks, the draining popliteal LN grewrapidly and became visibly enlarged, while the more distal inguinal LNbecame clearly palpable. When the tumors reached this point, there areno procedures that can be performed to alleviate discomfort and thesemice were euthanized. Consequently, overall survival cannot beadequately assessed in mice with massive tumor burden. Therefore, it wasdecided to assess two other parameters: recurrence free survival, wherespontaneous death or euthanized animals with excessive local recurrencetumor burden were scored as positive events; and the development of lungmetastases.

The combination of LOFU+IGRT provided a statistically significant(P=0.04) recurrence free survival advantage over either treatment alonein C57BL/6 mice (FIG. 6C). Notably, in all groups except C57BL/6LOFU+IGRT, local metastasis to the draining popliteal or inguinal LNfrequently necessitated the use of euthanasia. Furthermore, while micethat were treated with LOFU+IGRT showed a strict control of lungmetastases, in the other three groups, even animals with relativelylittle local recurrence ultimately died due to overwhelming lungmetastasis (FIG. 6D).

Discussion

The adaptive immune system constantly surveys for malignantlytransformed cells. This is largely achieved by recognition oftumor-associated antigens that prime the appropriate T cell repertoireto mount antitumor immune responses. However, tumors also employ diversemechanisms to evade the adaptive immune system and thwart antitumor Tcell responses (1). As a result, successful therapy against cancer hasto overcome the major obstacle of tumor-induced tolerance (36). Severalmechanisms have been described to explain how tumors induce tolerance indifferent T cell subtypes, including defective presentation of tumorantigens and inadequate activation of antigen presenting cells,signaling through co-inhibitory receptors, immunosuppression by factorsreleased within the tumor microenvironment and local recruitment ofsuppressor cells (9, 15-17, 20, 37-39). Treatments that promoteimmunogenic cell death (ICD) of cancer cells can mitigate and drivereversal of tolerance. The hallmarks of ICD include the release ofdamage-associated molecular patterns (DAMPs), translocation of certainchaperone complexes to the cell surface, and increased dendriticcell-mediated cross-presentation of tumor-associated antigens (40). Inthis study it was sought to investigate if novel treatment for melanomausing non-ablative LOFU would result in prevention, reversal ormitigation of tumor-induced tolerance and therefore in enhancedanti-tumor immune responses.

Thermally ablative HIFU, while able to control primary tumors, istypically not effective at preventing micro-metastatic invasions insurrounding or distant tissues, suggesting that cell death caused bythis FUS modality fails to adequately prime an adaptive anti-tumorimmune response. Indeed, local or distal micro-metastatic invasions maybe prevented or ameliorated by an adequately primed immune system thatcould eliminate the relatively small tumor load of cells that escapeinitial ablative treatment. In this study, using a B16 murine melanomamodel, we show that the use of non-ablative LOFU treatment enhances Tcell effector responses by overcoming the tolerizing effects of thetumor microenvironment and prevents local recurrence and distalmetastases when administered prior to an ablative treatment.

Development of T cell hyporesponsiveness to tumor antigens has beendescribed in T cells in several mouse tumor models and in human cancers(15, 18, 41). This laboratory has previously reported that tumor-antigenspecific CD4+ T cells become anergic in tumor bearing mice and express aseries of anergy-associated genes that have been shown to hinder theirability to proliferate and produce effector cytokines (21, 42).Furthermore, prevention of the expression of those genes in mice thatlack NFAT1 or Egr2, two transcription factors responsible for theexpression of anergy-inducing genes (43-45), leads to inhibition oftumor-antigen specific T cell hyporesponsiveness and improved control oflocal tumor growth (19, 21). Using two different B16 mouse melanomamodels, the data confirm that resident tumor antigen specific CD4+ Tcells in the tumor DLN upregulate the expression of anergy associatedgenes, including Grail, Itch, Cblb, Grg4, and Egr2. The activation ofthis program of gene expression was well correlated with a reducedability to produce cytokines following ex-vivo restimulation, supportingthat B16 melanoma induces an intrinsic state of hyporesponsiveness intumor antigen specific CD4+ T cells. Importantly, treatment of theprimary tumor with LOFU resulted in an increased ability of thosetumor-antigen specific CD4+ T cells to produce cytokine uponre-stimulation. US-induced restoration of the responsiveness to TCRengagement, in otherwise anergic cells, was accompanied by a reduction,to varying extents, of the expression of most anergy-inducing genes. Theabsence of FUS-induced changes in Foxp3 transcripts in DLN resident CD4+T cells in tumor bearing mice suggests, however, that FUS did not affectFoxp3+ Treg migration or differentiation and supports that LOFUprevented tumor-induced-tolerance by inhibiting T cell anergy.

Initial studies on tumor induced T cell anergy identified the key rolethat antigen presenting cells played in this process and defectivedendritic cell maturation has been defined as a major determinant ofinefficient priming of tumor-antigen specific T cells (20, 46).Recently, it has been shown that unstable immunological synapses formedbetween T cells and dendritic cells presenting tumor antigens result indelayed nuclear export of NFAT and the likely activation of atolerogenic NFAT-dependent program of gene expression that includes Egr2(47). Increased T cell activation that follows LOFU treatment of B16melanomas could potentially result from several different phenomena.First, treatment of the tumors with LOFU delivers both thermal andmechanical stress to the tumor cells. This stress could help generatenovel unique “non-self” tumor antigens that, in turn, could make thetumor more immunogenic and less able to induce tolerance. Alternativelyor additionally, the release of stress-induced danger signals by tumorcells could generate a tumor microenvironment that targeting dendriticcells would find less conducive to induction of tolerance in T cells.Stress associated molecular chaperones, including heat shock proteinsand calreticulin, have been implicated in dendritic cell maturation andenhanced anti-tumor immunity (32, 48-51). There is evidence that primarytumor lysates are rich in heat shock proteins that can triggermaturation signals in dendritic cells (52). Importantly, heat shockproteins are also capable of associating with and delivering antigenicpeptides from tumor cells to dendritic cells, furthermore their presencein the tumor cell plasma membrane has been associated with increasedimmune responses (53-55). Calreticulin has also been described to playan important role in antitumor response and its translocation to thesurface of tumor cells has been associated with increased phagocytosisof the cell by dendritic cells and immune activation (56, 57). Previousstudies using HIFU in murine adenocarcinoma models showed that thistreatment significantly increased expression of co-stimulatory moleculeson dendritic cells, which also produced higher levels of IL-12 andresulted in increased CTL activity (58, 59). The data show that LOFUinduces a redistribution of calreticulin in B16 cells and an increase inthe expression of the inducible heat shock protein Hsp70, suggestingthat cellular stress mediated by LOFU is capable of inducing changes inthe expression of those stress-induced proteins. Although no significantdifferences in the expression of MHC-II or B7 proteins were detected, itcannot be ruled out that LOFU may also induce other changes in dendriticcell function that can contribute to the potentiation of the efficientpresentation of tumor antigens. In any case, the data support that thethermal/mechanical stress inflicted by LOFU is likely responsible forthe enhanced tumor immunogenicity and for promoting T cell activationover anergy.

T cell tolerance induced by tumor antigens remains a major obstacle intreating cancer. Effective reversal of tolerance in tumor-specific Tcells is a key goal in clinical antitumor strategies. Our data find thatpre-established anergy in T cells can be reversed by lysates preparedfrom LOFU treated melanoma tumors. This observation opens up thepossibility that LOFU treatment of tumors could release novelimmunogenic molecules from tumor cells that would not only prevent butalso reverse pre-established tumor tolerance in T cells. Signalingthrough the IL-2 receptor has long been known to prevent and reverseclonal anergy in T cells (60-62). However, elevated amounts of IL-2 incould not be detected in any of lysates, untreated or treated with LOFU(data not shown), making presence of IL-2 an unlikely candidate to havecaused the reversal of anergy in the experiments. However, it ispossible that other factors could contribute to this phenotype. In fact,T cell co-stimulation through the TNFR family member OX-40 ligand hasalso been shown to prevent and overcome T cell anergy in addition toincreased effector response in both CD4+ and CD8+ T cells (63-65).Engagement of CD137, CD40 and blockade of PD1 have also been reported toprevent and reverse pre-established CD8+ T cell tolerance in vivo(66-68).

Pretreatment of melanoma tumors with LOFU before performing ablativetherapy using hypofractionated IGRT resulted in a significant delay intumor growth, and in several cases, complete regression of tumors wasobserved only in immunocompetent mice. Recurrence free survival in thesemice were also markedly improved following that protocol. In addition,incidences of lung metastases were minimal in mice that received LOFUprior to tumor ablation compared to mice that received only ablativeIGRT. Strikingly, the LOFU failed to confer similar protection whensimilar experiments were performed on T cell-deficient nude mice. Thisobservation indicates that the protective effect of LOFU is notrestricted only to control primary tumor, but can prevent theestablishment of metastatic foci either locally or distally. Thisprotection from metastasis could possibly result from theprevention/reversal or both of T cell tolerance to tumor antigens. LOFUpretreatment not only controlled tumor growth more effectively, but, asindicated before, likely resulted in the generation of stronglyimmunogenic IGRT-induced tumor death that provided protection frommetastasis and ensured longer recurrence free survival.

Prevention of T cell tolerance to endogenous tumor antigens is ofparamount importance in treating cancer. The work herein shows thattreatment of primary tumors with LOFU can accomplish that, making it acandidate therapy for the development of an in situ autologous tumorvaccine. FUS treatment of solid tumors, in combination with an ablativeapproach could prove to potentiate efficacy of primary tumoreradication, as well as prevention of metastases.

Methods and Materials

Mice: 6-8 week old C57BL/6, B6.Cg-Rag1tm1MomTyrp1B-wTg(TcraTcrb)9Rest/J(Tyrp1) and B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) mouse strains werepurchased from The Jackson Laboratory. BALBc/Nude mice were obtainedfrom National Cancer Institute, distributed through Charles River. Allmice were housed and maintained in pathogen-free facilities.

Culture of B16 cell lines and primary CD4+ T cells: B16-F1 and B16-F10melanoma cell lines were purchased from the American Type CultureCollection (ATCC). A highly aggressive subclone of B16-F10 (B16-M1) wasgenerated by isolating and expanding a metastatic clone that arose in aC57BL/6 mouse 6 weeks after surgical removal of an established primarytumor. The B16-OVA melanoma cell line was kindly provided by E. M. Lord(University of Rochester Medical Center, Rochester, N.Y.). Theexpression of OVA by B16-OVA cells was confirmed by real time PCR. Allmelanoma cells were cultured in DMEM (Thermo Scientific) supplementedwith 10% heat inactivated FBS, 2 mM L-Glutamine and 250 IU ofpenicillin/streptomycin.

CD4+ T cells were isolated using anti-CD4 conjugated magnetic Dynabeads(Life Technologies) according to the manufacturer's protocol. Whereindicated, CD4+ T cells were differentiated into TH1 helper cells byactivation with plate-bound anti-CD3Σ (clone 2C11; 0.25 (g/mL) andanti-CD28 (clone 37.51; 0.25 (g/mL) antibodies (BD Biosciences) andcultured for six days in DMEM supplemented with 10% heat inactivatedFBS, 2 mM L-glutamine, 50 (M 2-mercaptoethanol, nonessential amino acidsand essential vitamins (Cambrex), in the presence of murine IL-12 (10ng/mL) (eBioscience), anti-mouse IL-4 antibody (clone 11C.11; 10 μg/ml)and 10 U/mL recombinant human IL-2 (Biological Resources Branch of theNational Cancer Institute).

Tumor models: 3×10⁵ B16-F1 melanoma cells suspended in Hanks' BalancedSalt Solution (Invitrogen) were injected s.c. in the lumbar flanks ofmice. Melanoma tumors were induced in the footpads by injecting 2×10⁵B16-M1 cells in the dorsum of the right hind limb.

Tumor growth monitoring: Primary B16-M1 melanoma dorsal hind limb tumorswere measured three times per week with vernier calipers. Tumor volumewas calculated using an ellipsoid formula: V=(π/6×length×width×height).Primary dorsal hind limb tumors exhibit Gompertzian growth, with a phaseI volume of 30-50 mm³, phase II volume of 90-150 mm³, and phase IIIvolume of 300-500 mm³. Therefore, treatment efficacy was determined bydetermining the tumor growth delay (TGD) to 90-150 mm³, in which thetumor is in the exponential phase II. Tumors that reach 300-500 mm³begin to enter phase III due to anatomical and vascular limitations.Consequently, below-the-knee amputations were performed on mice withtumors ≥300-500 mm³, in accordance with IACUC approved protocol.

ELISA: 1.5 to 2.5×10⁴ T cells were left rested or stimulated with eitheranti-CD3Σ+anti-CD28 antibodies, T cell depleted OVA peptide 323-339(OVA323-339)-loaded splenocytes at a 1:5 T cell:splenocyte ratio, orCD11c+ purified dendritic cells (using CD11c-beads; Miltenyi Biotech)loaded with OVA323-339 or melanoma tumor lysates at a 1:3 dendriticcell:T cell ratio. Culture supernatants were typically harvested 24hours after stimulation, and IL-2 or IFNlevels were measured by asandwich ELISA (BD Biosciences).

Tumor lysates: Tumors were resected from tumor bearing mice, cut into1-2 mm pieces and passed through 40 μm nylon meshes. Cells were washedin PBS and resuspended in serum-free DMEM. Cell suspensions were thensnap frozen in liquid nitrogen, and thawed at 37° C. for five cycleswith visual confirmation of complete lysis by light microscopy. Thelysates were spun at 10,000 g for 15 minutes at 4° C., and the pelletswith cellular debris were discarded. The supernatant was used along withpurified dendritic cells to stimulate T cells.

Immunofluorescence staining tumor tissue was isolated, washed in PBS andembedded in OCT compound (Electron Microscopy Sciences). Tissue sections(5 m) were prepared and permeabilized with acetone for 5 min andincubated with goat serum for 30 min to block non-specificprotein-protein interactions. Tissue sections were incubated overnightwith the following antibodies: anti-Calreticulin (Pierce, PA5-25922),anti-Trp1 (Abcam, ab3312; clone TA99) and anti-Hsp70 (Novus Biologicals,NBP1-77455). Appropriate secondary antibodies were used for 30 min atroom temperature. DAPI (Invitrogen) was used to detect nuclei. At least10 fields/sample were blindly analyzed with an Inverted Olympus IX81fluorescence microscope.

Focused ultrasound therapy system. A therapy and imaging probe system(TIPS, Philips Research North America, Briarcliff Manor, N.Y., USA) wasutilized for all ultrasound exposures. The system is capable ofdelivering focused and spatiotemporally controlled ultrasound energy andconsists of a therapy control workstation, RF generators and controlelectronics, an 8-element spherical shell annular array ultrasoundtransducer (80 mm radius of curvature, 80 mm aperture), as well as amotion stage to allow for in-plane transducer movement and accuratepositioning perpendicular to ultrasound beam axis. The focusedultrasound beam can also be steered approximately +15 mm out-of-planeusing electronic deflection of the focal point. The ultrasound beampropagates vertically into the target through a thin (25 μm) circularplastic membrane, with acoustic coupling provided by degassed water.During therapy, the system allows adjustments of acoustic output power,ultrasound exposure duration, duty cycle, and ultrasound frequency.

In vivo focused ultrasound (FUS) therapy. Mice were anesthetized with acontinuous flow 1.5 liters/minute of 1.5% isoflurane in pure oxygen. Toensure proper acoustic coupling, the tumor-bearing leg or lumbar flankwere carefully shaved. Once the animal was positioned for therapy, thetumor was acoustically coupled to the TIPS system using degassed waterand ultrasound gel. The center of the tumor was then placed at the focallength of 80 mm from the transducer. Ultrasound exposures were deliveredto the tumor using a 1 mm grid pattern extending over the entire tumorvolume. Two layers of grid points (spaced 5 mm apart) were performed ineach tumor, resulting in approximately 160 discrete foci and 5 minexposure duration per tumor. The ultrasound transducer was operated at1.0 MHz, resulting in an ellipsoid focal spot approximately 1.5 mm indiameter and 12 mm in length (−6 dB of pressure), as measured along theellipsoid axes. Ultrasound exposures were delivered to the tumor using a1 mm grid pattern extending over the entire tumor volume. Prior totherapy, the tumor volume was measured to calculate the grid size forthe particular treatment. The duration of ultrasound exposure at eachgrid point was 1.5 s, after which the transducer was automaticallypositioned over the next grid point and the procedure repeated until theentire tumor volume was covered. Two layers of grid points wereperformed in each tumor. The therapeutic ultrasound device was operatedin continuous wave mode at a specific acoustic power/pressure regimen:acoustic power 3 W, peak negative pressure=2.93 MPa (80 mm focallength)/3.81 MPa (85 mm focal length); to provide non-ablativelow-energy FUS (LOFU). The resulting in situ intensity (Ispta) at thefocus was estimated to be 550 W/cm2 at a depth of 4 mm in tissue. Totalenergy deposition to a tumor was approximately 900 J.

In vivo hypofractionated cone beam CT image-guided Radiation Therapy(IGRT): All radiation was delivered using Xstrahl Limited's Small AnimalRadiation Research Platform (SARRP) to deliver a 10 Gy dose to a targettumor in 341 seconds. Anesthetized animals were placed on stage attachedto a motorized platform and the tumor-bearing right hind limb wasextended, elevated, and secured to a 1.5 cm adhesive platform tominimize extraneous tissue exposure. Once secure, a cone beam CT (CBCT)was performed and the data opened in 3D Slicer for tissue segmentationand treatment planning. 10 Gy each was delivered for three successivedays for a total hypofractionated dose of 30 Gy. In the combinationtherapy groups, LOFU was performed 2-4 hours prior to CBCT.

Pulmonary Metastasis Evaluation: Lungs were isolated from animals thatdied spontaneously, were euthanized or were sacrificed at the end of the8-week experiment. 1 mL of Fekete's solution (Ethanol, Glacial aceticacid and formaldehyde based bleaching fixative) was injected toinsufflate the lungs. The trachea was then clamped, and the entire lungsand heart removed en bloc and washed with PBS. The lungs were thenplaced in Fekete's solution and allowed to bleach for 48 hours prior toanalysis. The left lung and the 4 lobes of the right lung were isolatedand nodules counted with the aid of a dissecting microscope. Indistinctor fused nodules cannot be reliably enumerated; therefore, the lung waslabeled as too numerous to account and assigned an arbitrary metastasiscount of 250. Statistical analysis was performed using thenon-parametric Kruskal-Wallis test, followed by the Dunn's posttest formultiple comparisons.

Recurrence Free Survival: The following events were scored as positiveevents in our recurrence free survival analysis: spontaneous death withnecropsy validation of tumor involvement, euthanasia due to extensivelocal metastasis to the draining popliteal or inguinal lymph nodes, oreuthanasia due to moribund appearance indicating extensive systemictumor burden. The following non-tumor-dependent deaths were processed ascensored data: death within 24-48 hours of amputation or sacrifice ofany animals at the end of the 8-week experiment. In order to preventselective sacrifice of control or treated animals, cages were labeledusing an alphanumeric code such that animal institute veterinarians wereblinded from treatment and control groups. Recurrence free survival wasanalyzed using a Mantel-Cox test, with statistical significance definedas P<0.05.

Real time PCR: Total RNA was extracted from cells using RNeasy Micro kit(Qiagen), and cDNA was synthesized using qScript cDNA supermix (QuantaBiosciences). The cDNA samples were subjected to real time PCR usingPowerSYBR (Applied Biosystems) as the reporter dye on a StepOnePlus realtime PCR system (Applied Biosystems). Expression of the transcriptsstudied was normalized to beta actin. The primer sets used are thefollowing:

actinb: (SEQ ID NO: 1) F-GTGACGTTGACATCCGTAAAGA, (SEQ ID NO: 2)R-GCCGGACTCATCGTACTCC; Cblb: (SEQ ID NO: 3) F-GCAGCATCATTGACCCTTTCA,(SEQ ID NO: 4) R-ATGTGACTGGTGAGTTCTGCC; Grail: (SEQ ID NO: 5)F-ATGCAAGAGCTCAAAGCAGGAAGC, (SEQ ID NO: 6) R-GTGCGCAGCTGAAGCTTTCCAATA;Ikaros: (SEQ ID NO: 7) F-GCTGGCTCTCGGAGGAG, (SEQ ID NO: 8)R-CGCACTTGTACACCTTCAGC; Caspase3: (SEQ ID NO: 9) F-ACGCGCACAAGCTAGAATTT,(SEQ ID NO: 10) R-CTTTGCGTGGAAAGTGGAGT; Egr2: (SEQ ID NO: 11)F-TCAGTGGTTTTATGCACCAGC, (SEQ ID NO: 12) R-GAAGCTACTCGGATACGGGAG; Grg4:(SEQ ID NO: 13) F-TCACTCAAGTTTGCCCACTG, (SEQ ID NO: 14)R-CACAGCTAAGCACCGATGAG; Itch: (SEQ ID NO: 15) F-GTGTGGAGTCACCAGACCCT,(SEQ ID NO: 16) R-GCTTCTACTTGCAGCCCATC; Foxp3: (SEQ ID NO: 17)F-GGCCCTTCTCCAGGACAGA; (SEQ ID NO: 18) R-GCTGATCATGGCTGGGTTGT.

Flow cytometry: Cells were pre-blocked with Fc block (CD16/CD32)antibody prior to immunostaining. The following fluorochrome conjugatedantibodies were used: anti-B7.1, B7.2, CD11c, MHC-II, CD45, as well astheir respective isotype control antibodies (eBiosciences); anti-Hsp70(Novus Biologicals) and anti-TRP1 (Abcam). Dead cells were detected byusing a UV LIVE/DEAD R Fixable Dead Cell Stain Kit (Invitrogen). Theimmunostained cells were analyzed on an LSR-II Flow Cytometer (BectonDickinson), and post-acquisition analyses were carried out using theFlowJo software.

Example 2

The hypoxic tumor microenvironment generates oxidative EndoplasmicReticulum (ER) stress, resulting in protein misfolding and unfoldedprotein response (UPR). UPR induces several molecular chaperonesincluding heat-shock protein 90 (HSP90), which corrects proteinmisfolding and improves survival of cancer cells and resistance totumoricidal therapy although prolonged activation of UPR induces celldeath. The HSP90 inhibitor, 17AAG, has shown promise against varioussolid tumors, including prostate cancer (PC). However, therapeutic dosesof 17AAG elicit systemic toxicity. Herein a new paradigm is disclosedwhere the combination therapy of a non-ablative and non-invasive lowenergy focused ultrasound (LOFU) and a non-toxic, low dose 17AAG causessynthetic lethality and significant tumoricidal effects in mouse andhuman PC xenografts. LOFU induces ER stress and UPR in tumor cellswithout inducing cell death. Treatment with a non-toxic dose of 17AAGfurther increased ER stress in LOFU treated PC and switched UPR from acytoprotective to an apoptotic response in tumors resulting insignificant induction of apoptosis and tumor growth retardation.LOFU-induced ER stress makes the ultrasound-treated tumors moresusceptible to chemotherapeutic agents, such as 17AAG. LOFU-inducedchemosensitization is a novel therapy that can be used on tumors, forexample, locally advanced and recurrent tumors.

Treatment schema and toxicity of LOFU and 17AAG therapy: For each gridlocation, LOFU was administered for 1.5 seconds at 100% duty cycle,acoustic power of 3 W, and using ultrasound frequency of 1 MHz. Thisprotocol yielded an approximate in situ spatial-peak temporal-averageacoustic intensity of 270 W/cm², resulting in estimated averageintra-tumoral temperature elevation of 3.2° C. Post-treatment, therewere no signs of normal tissue toxicity such as alopecia, thermaldamage, or skin wounds. Preclinical pharmacokinetic studies in mice haveshown 17AAG to be widely distributed and to undergo extensive hepaticmetabolism. Systemic administration of 17AAG is known to be associatedwith significant hepatotoxicity, characterized by increases intransanminases and bile acids, and drug-related histopathologic lesionsin the gallbladder, common bile duct, and gastrointestinal tract.Therefore, we determined the dose of 17AAG that was nontoxic for ourtherapy. C57Bl/6 mice were treated with intraperitoneal injections of17AAG (25-75 mg/kg body weight) three times a week. Control mice wereinjected with equal volume of the vehicle DMSO, which was used tosolubilize 17AAG. Although higher doses of 17AAG (50-75 mg/kg of bodyweight) treatment achieved significant tumor growth retardation comparedto untreated control (untreated tumor, 1879±98.65 mm³ versus 17AAG 75mg/kg b.w., 485±24.25 mm3, p<0.003 and 50 mg/kg b.w., 964 mm3, p<0.007,respectively), Kaplan Meier survival analysis showed death in 50% ofmice after 21 days of treatment with a dose of 75 mg/kg of body weight.

A low dose of 17AAG that was found to be nontoxic was 25 mg/kg inC57Bl/6 mice and 14 mg/kg in Balb/c nude mice. Thus, these dose levelswere selected for the current study. The goal was to combine twotherapies that are nontoxic, albeit subtherapeutic, and examine whetherthe combination can be therapeutic. Combination treatment of LOFU+17AAGamplifies ER stress: Accumulation of misfolded proteins in the ERinduces a stress response with induction of chaperone proteins that helpin correction of protein misfolding. To detect the level of ER stress,the expression levels of ER chaperones, ERp44, ERp57, and ERp72 werequantitated among different treatment groups. ERp44 is responsible foroxidative protein folding [69]. ERp57 is an ER resident thiol disulfideoxidoreductase [70] while Erp72 is a disulfide isomerase. All theseproteins participate in the protein folding machinery of the ER.Compared to tumor tissues from animals that received no treatment orLOFU or 17AAG alone, immunoblot analysis demonstrated a significantincrease in the expression of ERp78 (p<0.03, FIGS. 7D & 7E), ERp44(p<0.05, FIGS. 7D & 7G), and ERp57 (p<0.04, FIGS. 7D & 7F) proteinlevels in tumor tissues following combination treatment with LOFU+17AAG.This suggests that 17AAG mediated inhibition of HSP90 may increase theunfolded protein burden in the ER, thereby prolonging ER stress.

LOFU+17AAG activates pro-apoptotic pathways of UPR and induces apoptosisin mouse and human prostate cancer tissues: ER stress activates thethree arms of UPR at the same time, thereby producing antagonisticcytoprotective and apoptotic signals at the same time. The fate of thecell depends upon the ability of its protein correction machinery tolower the ER stress, thereby attenuating the UPR. If ER stress persists,the cytoprotective pathways are eventually overwhelmed with the chronicactivation of PERK-mediated apoptotic pathways causing cellular demise.Since phosphorylation of PERK at Thr980 serves as a marker for itsactivation status, we performed immunoblot analysis that showed asignificant increase in pPERK levels in tumor tissue following treatmentwith 17AAG (FIG. 8A). Phosphorylated PERK levels were absent inuntreated and LOFU-treated tumors. However, combination treatment ofLOFLU+17AAG exhibited the highest levels of PERK phosphorylation (FIG.8A).

Since prolonged PERK activation attenuates protein synthesis in responseto ER stress through the phosphorylation of translation initiationfactor eIF2a at serine 51, the levels of phosphorylated eIF2α weredetermined. Treatment of RM1 tumors with 17AAG induced phosphorylationof eIF2α over the basal levels in untreated controls. LOFU treatmentresulted in marginal reduction of phosphorylated eIF2α levels. However,the highest levels of phosphorylated eIF2α were seen in tumors thatreceived combination treatment with LOFU+17AAG (FIG. 8B), corroboratingwith highest activation of PERK phosphorylation in these tumors comparedto other groups.

Although phosphorylated eIF2α decreases the translation of most cellularproteins, including pro-survival and anti-apoptotic proteins, itincreases the translation of a transcription factor, ATF4 that isresponsible for inducing the transcription of pro-apoptotic genes, suchas, CCAAT/enhancer-binding protein homologous protein (CHOP), therebypreparing the cell for programmed cell death in case the misfoldedproteins are not repaired and ER stress persists [71]. As expected, LOFUtreatment failed to induce CHOP levels (1.6±0.7 fold) over untreatedcontrols. In contrast, treatment with 17AAG alone induced CHOPtranscript levels to 14.8±2 fold, which was further increased to 25±1.3fold (p<0.006) in the combination treatment group of LOFU+17AAG,compared to untreated controls (FIG. 8C).

In order to examine whether downstream apoptotic genes are expressedfollowing CHOP induction by the combination therapy of LOFU+17AAG, amouse UPR qRT-PCR Array was used on total RNA isolated from tumortissues of various treatment groups. Heatmap analysis demonstrated thatpro-apoptotic target genes, such as Bax, Vcp, Pdia3, Armet, Ddit3,Mapk8, Mapk9, and Mapk0 were induced several folds following combinationtherapy with LOFU+17AAG compared to untreated controls (FIG. 8D). Therewas minimal induction of pro-apoptotic genes upon treatment with LOFUalone or 17AAG alone. This result indicates that the combination therapyof LOFU+17AAG activates PERK, induces CHOP, and switches on thepro-apoptotic pathway of the UPR. Indeed, TUNEL staining demonstratedthat LOFU induced minimal apoptosis over untreated controls. Treatmentwith 17AAG induced significant apoptosis in prostate tumors, which wasfurther increased by LOFU (p<0.004) (FIG. 8E). Thus, 17AAG-mediatedinhibition of HSP90 and activation of CHOP by the combination ofLOFU+17AAG switched on apoptotic cell death of prostate tumors.LOFU+17AAG inhibits Chaperone Mediated Autophagy (CMA) in tumor cells.

Degradation of misfolded proteins is mediated by the proteosomal pathwayand autophagy. Autophagy has been implicated in the tumorigenesisprocess in a context-dependent role, where it might provide amino acidsand other essential nutrients to the metabolic pathways of hypoxictumors that are nutrient deprived [72]. Indeed, an increase in CMAactivity has been described in a wide variety of human tumors and CMAhas been implicated in survival, proliferation, and metastases of tumorcells [73]. Therefore, the levels of two key proteins participating inautophagy were quantitated, Beclin, a marker of macroautophagy, andLAMP-2A lysosomal receptor, a marker of CMA in the tumor tissues ofvarious treatment cohorts. As shown in FIG. 10, Beclin levels remainunchanged with LOFU or 17AAG or the combination therapy (FIGS. 9A & 9C),indicating that macroautophagy was not altered with ultrasound therapy.However, LOFU alone or 17AAG alone induced the expression of LAMP-2A(FIGS. 9A & 9B), indicating a compensatory increase in CMA aftertherapies that increase the burden of misfolded proteins in the ER.Interestingly, the combination of LOFU+17AAG inhibited the levels ofLAMP-2A below the basal levels seen in these tumors. This suggests thatthe combination therapy reduces the growth of tumor cells and inducesapoptosis by increasing ER stress while suppressing CMA.

LOFU sensitizes human and murine prostate cancer grafts to non-toxic lowdoses of 17AAG: Treatment with LOFU alone or low dose of 17AAG (25 mg/kgbody weight) alone did not show any normal tissue toxic effect butfailed to inhibit tumor growth. However, combination therapy ofLOFU+17AAG reduced the growth of murine RM1 tumors (FIG. 10B). Theaverage estimated tumor growth is 5% (p<0.0001), 9% (p<0.0001) and 11%(p<0.0001) slower in LOFU, 17AAG and LOFU17AAG cohort compared tocontrol group. The median time to achieve tumor size 2000 mm³ incontrol, LOFU, and LOFU+17AAG were 18, 22, and 42 days, respectively.All the animals in 17AAG group achieved the size within the interval of26-30 days.

A similar degree of chemosensitization was observed in human PC3 tumorsin BalbC nu/nu mice upon application of LOFU together with low non-toxicdose of 17AAG (14 mg/kg of body weight), achieving significant tumorgrowth retardation (p<0.007) (FIG. 5C) without any immediate adverseside effects.

LOFU+17AAG treatment reduces the prostate cancer stem cell population intumor tissue: The effect of LOFU+17AAG-induced ER stress on PCstem/progenitor population was evaluated by flow cytometric analysis ofPC stem/progenitor cell surface markers [24, 25]. The percentage ofcells expressing cell surface SCA1 (FIGS. 11A & 11B) (p<0.004), CD44(FIGS. 11A & 11C) (p<0.003), CD133 (FIGS. 11A & 11D) (p<0.007), and α2β1integrin (p<0.005) (FIGS. 11A & 11E) was significantly decreased in thecombination treatment group, compared to control or single treatmentcohort. Mean fluorescence intensity (MFI) of all these markers remainedunaltered in all the three groups. qRT-PCR array of stem celltranscription factors demonstrated increase (>2 folds) in mRNA levels ofTIx3, Hoxa11, Pcna, Gli2, Runx1, Foxa2, Sp1, Tbx5, Hoxa10, Nfatc1,Gata6, and Notch2 (FIG. 11F), indicating that LOFU induces a PC steincell transcription signaling. Treatment with 17AAG also increased theexpression of some transcription factor mRNAs, such as FoxP1, Nrf2f, andPou5f1 that were present in LOFU-treated tumors. However, tumor treatedwith LOFU+17AAG down-regulated the expression of these genes, suggestingthat maximization of ER stress by the combination treatment might reducethe PC stem/progenitor cell population in tumors.

Discussion

The results demonstrate that the LOFU and chemotherapy combinationtherapy reprograms the expression of pro-apoptotic genes in tumors andinduces massive apoptosis in tumor xenografts, resulting in significanttumor growth retardation of mouse and human PC tumors. The effect ofLOFU can ameliorate resistance to a chemotherapy, and chemosensitizationcan be effected.

Methods and Materials

Animals

Five- to six weeks-old male C57Bl/6 (NCI-Fort Dietrich, Md., USA) miceand athymic nude (BalbC nu/nu mice, Jackson Laboratory, Bay Harbor, Me.,USA) mice were maintained ad libitum and all studies were performedunder the guidelines and protocols of the Institutional Animal Care andUse Committee of the Albert Einstein College of Medicine.

Tumor Model and Treatment

C57Bl/6 and BalbC nu/nu mice were injected subcutaneously with 1×10⁵RM-1 (murine prostate cancer cell line) and 1×10⁶ PC3 (human prostatecancer cell line) cells on the flank, respectively. Approximately 10days later, the tumor became palpable (3-5 mm in diameter), whereuponLOFU treatment was initiated. Mice were divided into 4 groups(n=5/group) receiving no treatment, LOFU, 17AAG (InvivoGen, San Diego,Calif., USA), and 17AAG+LOFU. Palpable tumors were treated with LOFUevery 3-4 days for five fractions administered over two weeks. Animalsreceived 17AAG three times a week during this time. Tumor volumemeasurements were performed twice weekly using Vernier calipers alongwith simultaneous physical assessment of signs of systemic toxicity(malaise and diarrhea).

LOFU System

A therapy and imaging probe system (TIPS, Philips Research NorthAmerica, Briarcliff Manor, N.Y., USA) was utilized for all ultrasoundexposures. The system includes an 8-element spherical shell annulararray transducer (80 mm radius of curvature, 80 mm aperture), as well asa motion stage to allow for transducer movement and accuratepositioning. The transducer was operated at 1.0 MHz, resulting in afocal spot approximately 1.5 mm in diameter and 12 mm in length (−6 dBof pressure). [12,13])

LOFU treatment protocol.

On treatment day, the animals were anesthetized with ketamine andxylazine (7:1 mg/ml for 100 l/mouse, i.p.). Once positioned for therapy,the tumor was acoustically coupled to the TIPS system using degassedwater and ultrasound gel.

Ultrasound exposure parameters were as follows: acoustic power of 3 Wand a duty cycle of 100%, yielding an approximate in situ spatial-peaktemporal-average intensity (Ispta) [74] of 270 W/cm² at a sonicationdepth of 3 mm in tissue, assuming an attenuation coefficient of 0.5 dBcm-1 MHz-1 [75]. Ultrasound exposures were delivered to the tumor usinga 2 mm grid pattern extending over the entire tumor volume. Prior toLOFU, the tumor volume was measured to calculate the grid size for theparticular treatment. The duration of LOFU exposure at each grid pointwas 1.5 s, after which the transducer was automatically positioned overthe next grid point and the procedure repeated until the entire tumorvolume was covered. This yielded a non-uniform energy delivery to thetumor.

In Vitro Temperature Rise Estimation.

Estimation of intra-tumoral temperature by invasive means couldundesirably modulate the therapeutic response of the combinationtreatment. Therefore, to estimate intra-tumoral temperature elevationusing the above described setup and therapy protocol, the ultrasoundexposures were performed in a 6 mm×6 mm area within a tissue-mimickingphantom, [76] into which a T-type thermocouple (diameter 200 μm) wasembedded at a depth of 3 mm. These in vitro exposures were repeated 5times and the results averaged.

Detection of Apoptosis In Situ

Apoptotic cells were detected in situ by performing TUNEL (TdT-mediateddigoxigenin labeled dUTP nick end labeling) staining. Briefly, paraffinembedded sections were de-paraffinized, rehydrated through gradedalcohols, and stained using an ApopTag kit (Intregen Co, Norcross, Ga.,USA). The apoptotic rate in tumor cells was quantified by counting thepercent of apoptotic cells in each high power field.

Immunoblot Analysis

24 hr post-LOFU the tumor cells were harvested, washed withphosphate-buffered saline, and lysed using TPER (Thermo FisherScientific, Rockford, Ill., USA). Cell lysates were subjected toSDS-PAGE, transferred to polyvinylidene difluoride membrane, andimmunoblotted with primary antibodies against PERK, pPERK, eIF2, peIF2,ERp72, ERp44, ERp57, Beclin (Cell signaling, Danvers, Mass., USA),Lamp2a (Abcam, Cambridge, Mass., USA), and horseradishper-oxidase-conjugated secondary antibody. The blots were developedusing the ECL kit (GE Healthcare, Piscataway, N.J., USA). Densitometricanalysis of immunoreactive bands of each blot was photographed and thenimages were digitized and analyzed by using Gel Doc XR system (Bio-Rad,Hercules, Calif., USA).

Real Time PCR analysis of UPR target genes 24 hr after LOFU treatmentthe RM1 tumor cells were lysed using RLT buffer mixed with 1%betamercaptoethanol from RNeasy Mini Kit (Qiagen, Valencia, Calif.,USA).

Qiagen's protocol for the RNeasy Mini Kit with on-column DNA digestionwas used to isolate RNA from the tumor lysates. The RNA samples werestored at −80° C., prior to further use. Isolated RNA was subjected tocDNA synthesis using the SuperScript™ First-Strand Synthesis System(Invitrogen, Grand Island, N.Y., USA). The splicing of XBP1 RNA wasdetected using the following primer pair 5′-ACTCGGTCTGGAAATCTG-3′ (SEQID NO:19) and 5′-TAGCCAGGAAACGTCTAC-3′ (SEQ ID NO:20) (FisherScientific, Pittsburgh, Pa., USA) [77]. Real time PCR was performed inLight Cycler real time PCR machine (Bio Rad Laboratories, Hercules,Calif., USA) using the Absolute QPCR SYBER Green Mix (ABgene, Rochester,N.Y., USA) according to the standard ABgene protocol. To check forprimer amplification specificity, a melting curve was generated at theend of the PCR and different samples containing the same primer pairshowed matching amplicon melting temperatures.

Primers used for real time PCR included GRP78 5′TTGCTTATGGCCTGGATAAGAGGG3′ (SEQ ID NO:21) and 5′TGTACCCTTGTCTTCAGCTGTCAC3′ (SEQ IDNO:22); EDEM 5′ TCATCCGAGTTCCAGAAAGCAGTC 3′ (SEQ ID NO:23) and 5′TTGACATAGAGTGGAGGGTCTCCT 3′ (SEQ ID NO:24) (Fisher Scientific). All theqRT-PCR and Real time PCR experiments were repeated three times. TheqRT-PCR and PCR array for apoptosis genes and stem cell transcriptionfactor were performed by SA Biosciences PCR array system (Frederick,Md., USA) according to manufacturer protocol. In brief, cDNA wereprepared from purified total RNA using RT² First Strand Kit (Qiagen)followed by PCR array using SA Bioscience PCR array kit. Data wasanalyzed by web based PCR array data analysis software from SABiosciences.

Flowcytometric Analysis

Flank tumors were treated with LOFU, 17AAG, and LOFU+17AAG in variouscohorts. 24 hours after treatment, tumor cells were isolated bycollagenase digestion and analyzed by flowcytometry for the expressionof prostate cancer stem cell markers, SCA1, CD44, and CD133. Isolatedtumor cells were stained with anti-SCA1 conjugated with FITC (BDBiosciences, La Jolla, Calif., USA), anti-CD133 conjugated with pacificblue (eBioscience, San Diego, Calif., USA) and anti-CD44 conjugated withPE (BD Biosciences, La Jolla, Calif., USA). Data acquisition wasperformed using LSRII (BD Biosciences) and analyzed by FlowJo v.7.1(Treestar Inc, Ashland, Oreg., USA) software.

Kaplan-Meier Survival Analysis

Mice survival/mortality in different treatment groups was analyzed byKaplan-Meier as a function of radiation dose using Sigma-Plot andGraphPad Prism (version 4.0 for OS X, San Diego, Calif., USA) software.

Statistical Analysis

For digital images, sampling regions were chosen at random for digitalacquisition for data quantitation. Digital image data was evaluated in ablinded fashion as to any treatment. A two-tailed Student's t-test wasused to determine significant differences (p<0.05) between experimentalcohorts with representative standard errors of the mean (SEM).

FIG. 12 shows an acoustic priming therapy (APT) device, generallydesignated by reference number 1, according to an exemplary embodimentof the present invention. The APT device 1 is powered by an electricalpower source (not shown) and includes a control system 10, amplifier 12,matching transformer 14 and transducer 16. To provide treatment, theultrasound transducer 16 may be positioned near or within a region ofthe patient's body 1000. A clinician may make appropriate adjustments tothe frequency and duration of the ultrasound pulses to be delivered bythe transducer 16 using a function generator at the control system 10.When the ultrasound transducer 16 is excited, a transmitting surface ofthe transducer element creates pressure waves in the bodily fluidssurrounding the ultrasound transducer 16. The pressure waves thenpropagate through the fluids and tissues within the patent's body 1000and ultimately reach the target region, thereby causing a non-ablative,sonic stress to the target tissue. As explained in further detailherein, the sonic stress delivered to the tissue may have manytherapeutic uses, and in the case of cancer treatment, for example, suchstress of cancer cells in a tumor may result in immunogenic modulation,radio-sensitization and chemo-sensitization. The ultrasound transducer16 may be repositioned to an adjacent area of the patient's body forfurther treatment.

The matching transformer 14 provides an impedance transformation betweenthe power supply and ultrasound transducer.

The amplifier 12 generates a transducer driver signal for driving thetransducer 16 based on the output signal of the control system 10. In anexemplary embodiment, the amplifier 12 may be a switched resonant poweramplifier, an example of which is disclosed in U.S. Pat. No. 7,396,336,the contents of which are incorporated herein by reference in theirentirety. In another embodiment, low impedance ultrasounddriver-transducer systems can be employed [78].

The transducer 16 generates acoustic power between 10 and 1000 W/cm²spatial peak temporal average intensity (I_(spta)) in a treatment zone.The ultrasound is applied continuously for a time in the range of from0.5 to 5 seconds, wherein the frequency is in the range of 0.01 to 10MHz. In some embodiments the minimum diameter of any ultrasound beam inthe treatment zone is about 1 cm.

In the embodiment shown in FIG. 12, the frequency of ultrasoundgenerated by the APT device 1 is in the range of about 10 KHz to about300 KHz. However, the APT device according to the present invention maygenerate higher frequencies such as, for example, frequencies in therange of about 300 KHz to about 3 MHz. As shown in FIG. 13, anembodiment of such an APT device, generally designated by referencenumber 100, may include a control system 110, amplifier 112, matchingtransformer 114 and transducer 116, such components having the samefunction and structure as previously described with reference to FIG.12. In embodiments, the transducer 116 may be a flat or concavepiston-type transducer comprised of single or multiple elements thatconvert another type of energy to acoustic energy.

As shown in FIG. 14, the APT device 1 and APT device 100 may beintegrated into a single system, generally designated by referencenumber 200, to provide improved efficacy and/or lower overall energyinput. The integrated system 200 provides focused low frequency andcollimated high frequency beams for APT treatment. In some embodimentsthe low frequency ultrasound is substantially focused according to whatis achievable for a given frequency or range of frequencies and the midfrequency is collimated. In some embodiments the transducer used toproduce the low frequency is concave and the transducer used to producethe mid frequency is planar.

As shown in FIGS. 15-17, the APT device 1, 100, 200 may operate inconjunction with an ultrasound monitoring system, generally designatedby reference 300. The ultrasound monitoring system 300 may be powered byan electrical power source (not shown) and includes a control system310, amplifier 312 and pulse receiver system 314. The ultrasoundmonitoring system 300 may be used to monitor and/or provide imaging ofthe target tissue prior to, during and/or after APT treatment, and inparticular the pulse receiver system 314 may include a transducer thatreceives pressure waves reflected from or generated by or from withinthe target tissue and the amplifier 312 generates electrical signalscorresponding to the received pressure waves. The control system 310generates output based on the electrical signals that can be used by aclinician to determine treatment status and/or other parameters.Although the ultrasound monitoring system 300 is shown as a separatecomponent from the APT device 1, 100, 200, it should be appreciated thatthe monitoring and APT delivery may be performed by a unitary system.

In some embodiments, the ultrasound monitoring system 300 is used toprovide information on the location of tissue to be treated. One ormore, non-therapeutic ultrasound transmit and receive sub-systems may beused to monitor APT treatment and the effects of treatment on tissues.

In some embodiments, the data collected and used for planning radiationtreatment are also used at least in part for planning ultrasoundtreatment.

In some embodiments, the data collected for APT treatment planning orAPT treatment is used in radiation treatment planning. In someembodiments the data collected for radiation treatment planning is usedfor APT treatment planning. In some embodiments the data collectedduring APT treatment is used in radiation treatment planning.

In some embodiments, ultrasound is applied at a lower frequency to treata particular location or locations identified in part by ultrasoundimaging performed at a higher frequency.

Various ultrasound-based imaging and monitoring modalities may be usedto monitor power deposition in tissues during APT treatment. In someembodiments, tissue temperature may be monitored via acoustic means[79].

In some embodiments, ultrasound elastography is used to monitortreatment. In some embodiments, harmonic imaging is used to monitortreatment. In some embodiments, thermal strain is measured. In someembodiments, the system is comprised of one or more ultrasound transmitand receive transducer sub-systems used to measure tissue strain. Tissuestrain information may be used to aim the treatment ultrasound beams toa desired tissue and, in some embodiments, prior to applying fulltreatment power to treatment transducers. For example, in oneembodiment, power is applied at 10 to 50% of the intended treatmentpower to one or more of the treatment transducers and strain of thetarget tissue is measured using ultrasound feedback. APT transducers maybe physically or electronically repositioned or more effectivelydirected to the target tissue based on strain imaging. In someembodiments, full power is applied from one or more transducers, but thetime is shorter than that used for therapeutic effect during the strainmeasurement period until a desired targeting is confirmed.

In some embodiments, thermometry is used to monitor treatment.

Each transducer of the APT system 1, 100, 200 may be a single transduceror may be an array of a plurality of transducers. FIG. 18 is aperspective view of a transducer, generally designated by referencenumber 400, according to an exemplary embodiment of the presentinvention. The transducer 400 includes an array of transducer elements402. Any number of transducer elements 402 may be sequentially arrangedalong the azimuth axis. The transducer elements 402 are supported on abacking block 404. Signal leads couple the electrode of each transducerelement 402 to transmit and receive circuitry as is well known. Thetransducer elements 402 convert electrical signals provided by thetransmit circuitry to pressure waves.

In some mid frequency embodiments (about 300 KHz to about 3 MHz), two ormore ultrasound transducers generate ultrasound beams that intersectwithin a treatment zone, herein denoted an intersection zone, with eachbeam having an I_(spta) in the intersection zone in the range of 10 to500 W/cm². In embodiments, two transducers generate ultrasound beamsthat intersect within a treatment zone, with each beam having anI_(spta) in the intersection zone in the range of 50 to 500 W/cm². Inembodiments, three transducers generate ultrasound beams that intersectwithin a treatment zone, with each beam having an I_(spta) in theintersection zone in the range of 50 to 500 W/cm².

In some embodiments, the plurality of beams are substantially in phasewith one another. In some embodiments, two ultrasound beams emanatingfrom separate ultrasound transducers are substantially in phase andintersect within a treatment zone, and each beam has an acoustic powerspatial peak intensity in the intersection zone in the range of 70 to100 W/cm² and the ultrasound is applied continuously from 1 to 5seconds. In some embodiments, three ultrasound beams emanating fromseparate ultrasound transducers are substantially of the same frequencyand in phase and intersect within a treatment zone, and each beam has anacoustic power spatial peak intensity in the intersection zone in therange of 50 to 70 W/cm² and the ultrasound is applied continuously for 1to 5 seconds.

In some embodiments, beams originating from separate transducers ortransducer elements each produce an I_(spta) of approximately 300 W/cm²in the treatment zone.

In some embodiments, at least one transducer diameter and the ultrasoundbeam emanating from this transducer is substantially larger than thetreatment zone. The use of one or more of such large transducer incombination with smaller transducers advantageously allows for lessprecise aiming of a high power, high volume beam while achievingeffective and faster treatments.

In some embodiments, an intense treatment zone is formed where two ormore beams cross paths, the intense treatment zone being equal to orgreater than about 1 cm perpendicular to the transmitted energydirection and also equal to or greater than about 1 cm parallel to thetransmitted direction.

In some embodiments, acoustic pressure applied to a treatment zone fromeach transducer is 0.1 to 10 MPa.

In some embodiments, the number of transducers that provide the intenseultrasound treatment zone is between 1 and 1000.

In some embodiments, one or more central frequencies are employed duringtreatment with central frequencies ranging from about 100 kHz to 20 MHz.

In some embodiments, the ultrasound from a given transducer is appliedcontinuously. In some embodiments the ultrasound emanating from a giventransducer is applied in pulses with repeating on time units and offtime units known as a duty cycle. The duty cycle may be in the range of1 on time units to 9 off time units.

In some embodiments, the transducers transmit single frequency tones ormulti-frequency chirps.

In some embodiments, the transducers are operated sequentially such thatthe total energy delivered to the target tissue for the entire course ofthe application is greater than that to surrounding tissues.

In some embodiments, the frequency is swept during application, in partto reduce undesirably high intensity zones in the zones near thetransducers.

In some embodiments, the transducer comprising treatment head ismechanically vibrated.

In some embodiments, the transducers are comprised of 2 dimensionalphased arrays, annular arrays and/or three-dimensional phased arrays.

In some embodiments, one or more ultrasound transducers are incorporatedinto one or more endoscopic devices.

The APT treatment systems disclosed herein have a low thermal dosecompared to thermal dosing schemes common in hyperthermic and ablativethermal therapies. In some embodiments, the maximum temperature reachedin the treatment zone is 45° C. during a treatment that lasts about 2seconds or less.

In consideration of thermal dose it is expected that therapeutic effectis obtained through a thermal mechanism. While not wishing to be boundby theory, mechanical effects coupled with thermal effects may explainin part observed efficacy of treatments using disclosed devices, systemsand methods.

In some embodiments, coupling media is used between the transducers andthe patient's body to efficiently transmit ultrasound waves and in someembodiments to provide a desired distance between a transducer and atreatment zone. In some embodiments, the coupling media is circulated tocool the transducer or the patient's body during treatment or both.Separate fluids may be used for purposes of transmitting ultrasound,providing spacing and providing cooling to the patient and systemcomponents.

While not bound by theory, APT treatment using the systems describedherein can promote the interactions between cells, and between cells andmatrix proteins. Interactions between cancer cells and immune cells andinteractions between immune cells, for example T Cells and DCs.

In some embodiments, APT treatment may disrupt protein complexes, forexample protein folding complexes.

For patients with diseases that benefit from treatment with multiplemodalities, permeation of entire targeted treatment zone and lesionswithin these zones, ease of application and short duration treatments ofeach modality is desirable.

Various other aspects of the APT treatment device and APT treatmentmodalities according to exemplary embodiments of the present inventionwill now be described:

Positioning Apparatus

In some embodiments, transducer applicators are designed so that theymay be hand-held by the clinician or care giver. In some embodiments,applicators are mounted to a mechanical positioning device, such as thepositioning device 500 illustrated in FIG. 19. The positioning device500 may be manually manipulated or robotic controlled. In the presentembodiment, the positioning device 500 is an arc-shaped rail on whichthe transducer travels, and in particular the transducer may be attachedto a cable-driven carriage that is in turn mounted on the rail. The railitself may be rotatable so that the transducer can be positioned inthree dimensions. The positioning device 500 may be large enough that apatient can fit underneath and within the target range of thetransducer. Although FIG. 19 shows only one transducer positioned on therail, it should be appreciated that more than one transducer may bedisposed on the rail and/or other rails may be provided that support oneor more other transducers. In some embodiments, a stewart platform,sometimes referred to as a hexapod, may be used in the positioningapparatus. Computer programming may be used to set treatment parametersand operate positioning apparatuses.

Equipment and Patient Cooling

In some embodiments, the treatment system comprises patient coolingmechanisms to cool the skin exposed to ultrasound energy or otherenergy.

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What is claimed is:
 1. An acoustic priming therapy device comprising: acontrol system; and one or more transducers coupled to the controlsystem and configured to produce one or more ultrasound beams such thata waveform of the one or more ultrasound beams has a spatial peaktemporal average acoustic output intensity (I_(spta)) of between 1 and1000 W/cm² in a treatment zone, wherein the one or more ultrasound beamsare applied for 0.5 to 5 seconds, wherein the one or more transducersproduce one or more ultrasound frequencies in the range of 0.01 to 10MHz, and wherein the one or more transducers comprise at least twotransducers that are configured to produce columnated ultrasound suchthat the beam profile waist at −3 dB is not less than 5 mm in thetreatment zone.
 2. The device of claim 1, wherein each of the one ormore transducers is configured to produce one or more ultrasoundfrequencies in the range of 0.05 to 5 MHz or 0.5 to 1.5 MHz; and anI_(spta) of between 20 and 1000 W/cm².
 3. The device of claim 1, whereina transducer of the one or more transducers comprises a singletransducer element or multiple transducer elements.
 4. The device ofclaim 3, wherein the transducer comprises two or more transducers ortransducer elements configured to produce two or more frequencies. 5.The device of claim 4, wherein the two or more frequencies comprise afirst ultrasound frequency within the range of 300 kHz to 3 MHz producedby one of the two or more transducers or transducer elements and asecond ultrasound frequency within the range of 30 kHz to 300 kHzproduced by one of the two or more transducers or transducer elements.6. The device of claim 1, wherein the one or more transducers comprisetwo or more ultrasound transducers that generate ultrasound beams thatpass through the treatment zone, with each beam having an I_(spta) inthe treatment zone in the range of 10 to 500 W/cm².
 7. The device ofclaim 1, wherein the one or more ultrasound beams produced by the one ormore transducers is applied to the treatment zone of at least one cubiccentimeter of tumor.
 8. The device of claim 1, wherein the one or moretransducers comprise two transducers or three transducers that generateultrasound beams that are substantially in phase and intersect within atreatment zone, wherein each beam has an I_(spta) in the intersectionzone in the range of 70 to 100 W/cm², and wherein the ultrasound isapplied continuously from 1 to 5 seconds per treatment zone.
 9. Thedevice of claim 1, wherein the one or more transducers is configured toproduce the one or more ultrasound beams for application to two or morevolumes of a tumor over a period of time of less than one hour.
 10. Thedevice of claim 1, wherein each of the one or more transducers isconfigured to produce the one or more ultrasound beams for applying anacoustic pressure to the treatment zone of 0.1 to 10 MPa and wherein theone or more transducers is configured with a duty cycle of 10, 20, 30,40, 50, 60, 70, 80, 90%, or 100%.
 11. The device of claim 1, wherein theone or more transducers are configured to produce the one or moreultrasound beams in single frequency tones or multi-frequency chirps.12. The device of claim 1, wherein the one or more transducers areconfigured to produce the one or more ultrasound beams that raises atemperature in the treatment zone to less than 45° C.
 13. The device ofclaim 1, wherein the one or more transducers are configured to create anacoustic pressure in the treatment zone to induce a non-ablative stressin a tumor in the treatment zone.
 14. The device of claim 1, wherein theone or more transducers are configured to produce the one or moreultrasound beams have energy and intensity to prime immune system cellsfor a chemotherapeutic agent, wherein the energy and intensity fallbetween energies and intensities that induce ablative effects and thathave diagnostic effects.
 15. The device of claim 1, wherein the one ormore transducers comprise two or more transducers configured to operatesequentially in time and produce a low thermal dose and a mechanicalvibration, wherein the thermal dose comprises a maximum temperature of45° C. in the treatment zone.